Method of inhibiting hepatic fibrosis as a result of liver replacement or repair using A2B adenosine receptor antagonists

ABSTRACT

The invention is related to methods of inhibiting hepatic fibrosis using A 2B  adenosine receptor antagonists and utility in the treatment and prevention of liver damage caused by alcohol abuse, surgical intervention, viral hepatitis, the ingestion of hepatotoxic drugs, or other hepatic diseases. The invention also relates to pharmaceutical compositions for use in the method.

This application is a continuation of U.S. patent application Ser. No.12/853,510, filed Aug. 10, 2010, which is a divisional of U.S. patentapplication Ser. No. 11/687,236, filed Mar. 16, 2007, now U.S. Pat. No.7,795,268, which claims priority to U.S. Provisional Patent ApplicationNo. 60/783,575, filed Mar. 17, 2006, the entirety of which areincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 24, 2013, isnamed 045710-5603_SL.bd and is 1,374 bytes in size.

FIELD OF THE INVENTION

The present invention relates to methods of preventing and treatinghepatic disease using A_(2B) adenosine receptor antagonists. Thisinvention finds utility in the treatment and prevention of liver damagecaused by alcohol abuse, surgical intervention, viral hepatitis, theingestion of hepatotoxic drugs, or other hepatic diseases. The inventionalso relates to pharmaceutical compositions for use in the method.

BACKGROUND

Hepatic disease can take a wide variety of forms including, but notlimited to, necrosis, steatosis, fibrosis, and cholestatis. Other formsof liver disease can result from the ingestion of hepatotoxic medicinessuch as chemotherapy and cancer drugs, antibiotics, analgesics,antiemetics, and other medications. Also, alcohol and drug abuse arewell known causes of liver disease. Typical causes of hepatic diseaseinclude, but are not limited to, viral and alcoholic hepatitis, Wilson'sdisease, hemochromatosis, steatosis, and nonalcoholic steatohepatitis(NASH).

Hepatic fibrosis is a common aspect of many, if not all, hepaticdiseases and is defined as the formation of scar tissue in the liver.The scarring develops as the liver attempts to repair cellular damageinduced by the ingestion of hepatotoxins, as a consequence of chronicliver inflammation, or as a consequence of physical insult. Hepaticfibrosis may also result as a consequence of surgical intervention andhepatotoxic drug therapy, i.e., liver replacement or repair orchemotherapy. In many cases, hepatic fibrosis produces permanentscarring of the hepatic tissue, a condition commonly referred to ascirrhosis.

Recent studies have disclosed that adenosine plays a role in thedevelopment and progression of hepatic fibrosis. Chunn et al. (2006) Am.J. Physiol Lung Cell Mol Physiol, 290(3):L579-87, detected increasedhepatic fibrosis in adenosine deaminase (ADA) deficient mice. The miceutilized by Chunn et al. are genetically engineered to possess partialADA enzyme activity and thereby accumulate adenosine of a prolongedperiod of time.

Adenosine is a naturally occurring nucleoside, which exerts itsbiological effects by interacting with a family of adenosine receptorsknown as A₁, A_(2A), A_(2B), and A₃, all of which modulate importantphysiological processes. Of the various receptors, A_(2B) adenosinereceptors have been shown to modulate the synthesis and release ofangiogenic factors and inflammatory cytokine and chemokines and arebelieved to be most significantly involved in inflammatory conditionsvia their connection to mast cell activation, vasodilation, andregulation of cell growth (See Adenosine A_(2B) Receptors as TherapeuticTargets, Drug Dev Res 45:198; Feoktistov et al., Trends Pharmacol Sci19:148-153).

Surprisingly, it has now been found that A_(2B) adenosine receptorantagonists are also useful in the prevention and treatment of hepaticdisease. Accordingly, it is desired to provide a method of treatingand/or preventing hepatic disease by administration of compounds thatare potent, fully or partially selective, A_(2B) antagonists, i.e.,compounds that inhibit the A_(2B) adenosine receptor.

SUMMARY OF THE INVENTION

In one embodiment of the invention, a method is provided for thetreatment and prevention of hepatic disease by administration of atherapeutically effective amount of an A_(2B) adenosine receptorantagonist to a mammal in need thereof. The hepatic disease may take theform of necrosis, fibrosis, cholestatis, cirrhosis, viral and alcoholichepatitis, Wilson's disease, hemochromatosis, steatosis, andnonalcoholic steatohepatitis (NASH) or the hepatic disease may be theconsequence of surgical intervention or drug therapy with a hepatoxicagent, i.e., liver replacement or repair or chemotherapy.

In a second embodiment of the invention, a method is provided fordecreasing the hepatotoxic side effects of chemotherapy or radiation byadministration of a therapeutically effective amount of an A_(2B)adenosine receptor antagonist to a mammal undergoing such treatment.

In another embodiment of the invention, a method is provided for thetreatment and prevention of hepatic disease by administration to amammal in need thereof, a therapeutically effective amount of an A_(2B)adenosine receptor antagonist having the structure of Formula I orFormula II:

wherein:

-   R¹ and R² are independently chosen from hydrogen, optionally    substituted alkyl, or a group -D-E, in which D is a covalent bond or    alkylene, and E is optionally substituted alkoxy, optionally    substituted cycloalkyl, optionally substituted aryl, optionally    substituted heteroaryl, optionally substituted heterocyclyl,    optionally substituted alkenyl or optionally substituted alkynyl,    with the proviso that when D is a covalent bond E cannot be alkoxy;-   R³ is hydrogen, optionally substituted alkyl or optionally    substituted cycloalkyl;-   X is optionally substituted arylene or optionally substituted    heteroarylene;-   Y is a covalent bond or alkylene in which one carbon atom can be    optionally replaced by —O—, —S—, or —NH—, and is optionally    substituted by hydroxy, alkoxy, optionally substituted amino, or    —COR, in which R is hydroxy, alkoxy or amino; and-   Z is optionally substituted monocyclic aryl or optionally    substituted monocyclic heteroaryl; or-   Z is hydrogen when X is optionally substituted heteroarylene and Y    is a covalent bond.

In yet another embodiment of the invention, pharmaceutical formulationsare provided, comprising a therapeutically effective amount of an A_(2B)adenosine receptor antagonist, and at least one pharmaceuticallyacceptable carrier. The formulation is preferably for oraladministration.

One preferred group of compounds of Formula I and II are those in whichR¹ and R² are independently hydrogen, optionally substituted loweralkyl, or a group -D-E, in which D is a covalent bond or alkylene, and Eis optionally substituted phenyl, optionally substituted cycloalkyl,optionally substituted alkenyl, or optionally substituted alkynyl,particularly those in which R³ is hydrogen.

Within this group, a first preferred class of compounds include those inwhich R¹ and R² are independently lower alkyl optionally substituted bycycloalkyl, preferably n-propyl, and X is optionally substitutedphenylene. Within this class, a preferred subclass of compounds arethose in which Y is alkylene, including alkylene in which a carbon atomis replaced by oxygen, preferably —O—CH₂—, more especially where theoxygen is the point of attachment to phenylene. Within this subclass, itis preferred that Z is optionally substituted oxadiazole, particularlyoptionally substituted [1,2,4]-oxadiazol-3-yl, especially[1,2,4]-oxadiazol-3-yl substituted by optionally substituted phenyl oroptionally substituted pyridyl.

A second preferred class of compounds include those in which X isoptionally substituted 1,4-pyrazolene. Within this class, a preferredsubclass of compounds are those in which Y is a covalent bond oralkylene, especially lower alkylene, and Z is hydrogen, optionallysubstituted phenyl, optionally substituted pyridyl, or optionallysubstituted oxadiazole. Within this subclass, one preferred embodimentincludes compounds in which R¹ is lower alkyl optionally substituted bycycloalkyl, and R² is hydrogen. A more preferred embodiment includesthose compounds in which Y is —(CH₂)— or —CH(CH₃)— and Z is optionallysubstituted phenyl, or Y is —(CH₂)— or —CH(CH₃)— and Z is optionallysubstituted oxadiazole, particularly 3,5-[1,2,4]-oxadiazole, or Y is—(CH₂)— or —CH(CH₃)— and Z is optionally substituted pyridyl. Withinthis subclass, also preferred are those compounds in which R¹ and R² areindependently lower alkyl optionally substituted by cycloalkyl,especially n-propyl. More preferred are those compounds in which Y is acovalent bond, —(CH₂)— or —CH(CH₃)— and Z is hydrogen, optionallysubstituted phenyl, or optionally substituted pyridyl, particularlywhere Y is a covalent bond and Z is hydrogen.

An additional subgroup of preferred compounds are those in which R³ is asubstituted alkyl group of the formula —CHR⁴OR⁵ so that the compoundshave the structure of Formula III:

wherein:

-   R⁴ is hydrogen or methyl; and-   R¹ is —C(O)R, in which R is independently optionally substituted    lower alkyl, optionally substituted aryl, or optionally substituted    heteroaryl; or-   R⁵ is —P(O)(OR⁶)₂, in which R⁶ is hydrogen or lower alkyl optionally    substituted by phenyl or heteroaryl;    and the pharmaceutically acceptable salts thereof.

At present, the preferred compounds for use in the invention include,but are not limited to:

-   1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]-methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione;-   1-propyl-8-[1-benzylpyrazol-4-yl]-1,3,7-trihydropurine-2,6-dione;-   1-butyl-8-(1-{[3-fluorophenyl]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione;-   1-propyl-8-[1-(phenylethyl)pyrazol-4-yl]-1,3,7-trihydropurine-2,6-dione;-   8-(1-{[5-(4-chlorophenyl)(1,2,4-oxadiazol-3-yl)]methyl}pyrazol-4-yl)-1-propyl-1,3,7-trihydropurine-2,6-dione;-   8-(1-{[5-(4-chlorophenyl)(1,2,4-oxadiazol-3-yl)]methyl}pyrazol-4-yl)-1-butyl-1,3,7-trihydropurine-2,6-dione;-   1,3-dipropyl-8-pyrazol-4-yl-1,3,7-trihydropurine-2,6-dione;-   1-methyl-3-sec-butyl-8-pyrazol-4-yl-1,3,7-trihydropurine-2,6-dione;-   1-cyclopropylmethyl-3-methyl-8-{1-[(3-trifluoromethylphenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   1,3-dimethyl-8-{1-[(3-fluorophenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   3-methyl-1-propyl-8-{1-[(3-trifluoromethylphenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   3-ethyl-1-propyl-8-{1-[(3-trifluoromethylphenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   1,3-dipropyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione;-   1,3-dipropyl-8-{1-[(3-fluorophenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   1-ethyl-3-methyl-8-{1-[(3-fluorophenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   1,3-dipropyl-8-{1-[(2-methoxyphenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   1,3-dipropyl-8-(1-{[3-(trifluoromethyl)-phenyl]ethyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione;-   1,3-dipropyl-8-{1-[(4-carboxyphenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   2-[4-(2,6-dioxo-1,3-dipropyl(1,3,7-trihydropurin-8-yl))pyrazolyl]-2-phenylacetic    acid;-   8-{4-[5-(2-methoxyphenyl)-[1,2,4]oxadiazol-3-ylmethoxy]phenyl}-1,3-dipropyl-1,3,7-trihydropurine-2,6-dione;-   8-(4-[5-(3-methoxyphenyl)-[1,2,4]oxadiazol-3-ylmethoxy]phenyl-1,3-dipropyl-1,3,7-trihydropurine-2,6-dione;-   8-{4-[5-(4-fluorophenyl)-[1,2,4]oxadiazol-3    ethoxy]phenyl}-1,3-dipropyl-1,3,7-trihydropurine-2,6-dione.-   1-(cyclopropylmethyl)-8-[1-(2-pyridylmethyl)pyrazol-4-yl]-1,3,7-trihydropurine-2,6-dione;-   1-n-butyl-8-[1-(6-trifluoromethylpyridin-3-ylmethyl)pyrazol-4-yl]-1,3,7-trihydropurine-2,6-dione;-   8-(1-{[3-(4-chlorophenyl)(1,2,4-oxadiazol-5-yl)]methyl}pyrazol-4-yl)-1,3-dipropyl-1,3,7-trihydropurine-2,6-dione;-   1,3-dipropyl-8-[1-({5-[4-(trifluoromethyl)phenyl]isoxazol-3-yl}methyl)pyrazol-4-yl]-1,3,7-trihydropurine-2,6-dione;-   1,3-dipropyl-8-[1-(2-pyridylmethyl)pyrazol-4-yl]-1,3,7-trihydropurine-2,6-dione;-   3-{[4-(2,6-dioxo-1,3-dipropyl-1,3,7-trihydropurin-8-yl)pyrazolyl]methyl}benzoic    acid;-   1,3-dipropyl-8-(1-{[6-(trifluoromethyl)(3-pyridyl)]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione;-   1,3-dipropyl-8-{1-[(3-(1H-1,2,3,4-tetraazol-5-yl)phenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   6-{[4-(2,6-dioxo-1,3-dipropyl-1,3,7-trihydropurin-8-yl)pyrazolyl]methyl}pyridine-2-carboxylic    acid;-   3-ethyl-1-propyl-8-[1-(2-pyridylmethyl)pyrazol-4-yl]-1,3,7-trihydropurine-2,6-dione;-   8-(1-{[5-(4-chlorophenyl)isoxazol-3-yl]methyl}pyrazol-4-yl)-3-ethyl-1-propyl-1,3,7-trihydropurine-2,6-dione;-   8-(1-{[3-(4-chlorophenyl)(1,2,4-oxadiazol-5-yl)]methyl}pyrazol-4-yl)-3-ethyl-1-propyl-1,3,7-trihydropurine-2,6-dione;-   3-ethyl-1-propyl-8-(1-{[6-(trifluoromethyl)(3-pyridyl)]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione;-   1-(cyclopropylmethyl)-3-ethyl-8-(1-{[6-(trifluoromethyl)(3-pyridyl)]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione;-   3-ethyl-1-(2-methylpropyl)-8-(1-{[6-(trifluoromethyl)(3-pyridyl)]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione;-   [3-ethyl-2,6-dioxo-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)-1,3,7-trihydropurin-7-yl]methyl    acetate;-   [3-ethyl-2,6-dioxo-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)-1,3,7-trihydropurin-7-yl]methyl    2,2-dimethylpropanoate;-   [3-ethyl-2,6-dioxo-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)-1,3,7-trihydropurin-7-yl]methyl    butanoate; and-   [3-ethyl-2,6-dioxo-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}-pyrazol-4-yl)(1,3,7-trihydropurin-7-yl)]methyl    dihydrogen phosphate.

SUMMARY OF THE FIGURES

FIG. 1 graphically illustrates the mRNA levels of AdoR subtypes inHHSCs. Total RNA isolated from HHSCs was subjected to real-time RT-PCRanalysis. The relative levels of the AdoR transcripts are presented aspercentages of the β-actin transcript. Data shown are averages ±SEM(n=4). nd denotes not detected.

FIG. 2 shows the effects of AdoR agonists and antagonist on cellularcAMP accumulation in HHSCs. (A) Concentration-response curves ofCGS-21680 (CGS, circle) and NECA in the absence (square) or presence(triangle) of the A_(2B) receptor antagonist8-(1-{[5-(4-chlorophenyl)(1,2,4-oxadiazol-3-yl)]methyl}pyrazol-4-yl)-1-propyl-1,3,7-trihydropurine-2,6-dione(compound (1)) (1 μM). (B) Lack of effect of CPA (1 μM) and IB-MECA (1M,1 μM) on forskolin (Fsk, 10 μM)-induced cellular cAMP accumulation. Datashown are averages ±SEM (n=6 in A and n=5 in B).

FIG. 3 graphs the effects of adenosine (A) and NECA (B) on the releaseof IL-6 by HHSCs. Cells were treated with vehicle, adenosine and NECA inthe absence or presence of compound (1) for 24 h. Media from treatedcells were collected, and the concentrations of IL-6 were determinedusing ELISA. Data shown are the averages ±SEM (n=3). *: p<0.05, comparedto control; #: p<0.05, compared to NECA (10 μM)-treated cells in B.

FIG. 4 shows the effects of NECA on the mRNA levels of α-smooth muscleactin (A) and α-1 pro-collagen (B) determined using real-time RT-PCR.HHSCs were incubated with NECA (10 μM) for 1 h. Cells incubated withvehicle were used as control. The expression levels of the target mRNAwere normalized to that of β-actin. Data shown are averages ±SEM (n=4 inA, and n=5 in B). *: p<0.05, compared to control.

FIG. 5 illustrates the effects of NECA on collagen production by HHSCs.Cells were treated with vehicle, NECA in the absence or presence ofcompound (1) or anti-IL-6 antibody for 24 h. Media from treated cellswere collected, and the concentrations of collagen were determined usingSircol collagen assay. Data shown are the averages ±SEM (n=4-6). *:p<0.05, compared to control; #: p<0.05, compared to NECA (10 μM)-treatedcells.

FIG. 6 illustrates the effects of A_(2B) receptor antagonism on plasmaAST levels in ADA^(−/−) mice. Mouse plasma was collected in EDTA, andthe activity of AST was determined using Infinity™ AST assay. Data shownare the averages ±SEM (n=6-8). *: p<0.05, compared to ADA⁺ treated withvehicle (ADA+V); #: p<0.05, compared to ADA^(−/−) mice treated withvehicle (ADA−/−V).

DETAILED DESCRIPTION OF THE INVENTION Definitions and General Parameters

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. This term isexemplified by groups such as methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, t-butyl, n-hexyl, n-decyl, tetradecyl, and the like.

The term “substituted alkyl” refers to:

-   1) an alkyl group as defined above, having 1, 2, 3, 4 or 5    substituents, preferably 1 to 3 substituents, selected from the    group consisting of alkenyl, alkynyl, alkoxy, cycloalkyl,    cycloalkenyl, acyl, acylamino, aryloxy, amino, aminocarbonyl,    alkoxycarbonylamino, azido, cyano, halogen, hydroxy, keto,    thiocarbonyl, carboxy, carboxyalkyl, arylthio, heteroarylthio,    heterocyclylthio, thiol, alkylthio, aryl, aryloxy, heteroaryl,    aminosulfonyl, aminocarbonylamino, heteroaryloxy, heterocyclyl,    heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,    —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, SO₂-aryl and —SO₂-heteroaryl.    Unless otherwise constrained by the definition, all substituents may    optionally be further substituted by 1, 2, or 3 substituents chosen    from alkyl, carboxy, carboxyalkyl, aminocarbonyl, hydroxy, alkoxy,    halogen, CF₃, amino, substituted amino, cyano, and —S(O)_(n)R, where    R is alkyl, aryl, or heteroaryl and n is 0, 1 or 2; or-   2) an alkyl group as defined above that is interrupted by 1-10 atoms    independently chosen from oxygen, sulfur and NR_(a)—, where R_(a) is    chosen from hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl,    alkynyl, aryl, heteroaryl and heterocyclyl. All substituents may be    optionally further substituted by alkyl, alkoxy, halogen, CF₃,    amino, substituted amino, cyano, or —S(O)_(n)R, in which R is alkyl,    aryl, or heteroaryl and n is 0, 1 or 2; or-   3) an alkyl group as defined above that has both 1, 2, 3, 4 or 5    substituents as defined above and is also interrupted by 1-10 atoms    as defined above.

The term “lower alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain having 1, 2, 3, 4, 5, or 6 carbon atoms.This term is exemplified by groups such as methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, t-butyl, n-hexyl, and the like.

The term “substituted lower alkyl” refers to lower alkyl as definedabove having 1 to 5 substituents, preferably 1, 2, or 3 substituents, asdefined for substituted alkyl, or a lower alkyl group as defined abovethat is interrupted by 1, 2, 3, 4, or 5 atoms as defined for substitutedalkyl, or a lower alkyl group as defined above that has both 1, 2, 3, 4or 5 substituents as defined above and is also interrupted by 1, 2, 3,4, or 5 atoms as defined above.

The term “alkylene” refers to a diradical of a branched or unbranchedsaturated hydrocarbon chain, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms, preferably 1-10carbon atoms, more preferably 1, 2, 3, 4, 5 or 6 carbon atoms. This termis exemplified by groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—),the propylene isomers (e.g., —CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

The term “lower alkylene” refers to a diradical of a branched orunbranched saturated hydrocarbon chain, preferably having from 1, 2, 3,4, 5, or 6 carbon atoms.

The term “lower alkylene” refers to a diradical of a branched orunbranched saturated hydrocarbon chain, preferably having from 1, 2, 3,4, 5, or 6 carbon atoms.

The term“substituted alkylene” refers to:

-   (1) an alkylene group as defined above having 1, 2, 3, 4, or 5    substituents selected from the group consisting of alkyl, alkenyl,    alkynyl, alkoxy, cycloalkyl, cycloalkenyl, acyl, acylamino, acyloxy,    amino, aminocarbonyl, alkoxycarbonylamino, azido, cyano, halogen,    hydroxy, keto, thiocarbonyl, carboxy, carboxyalkyl, arylthio,    heteroarylthio, heterocyclylthio, thiol, alkylthio, aryl, aryloxy,    heteroaryl, aminosulfonyl, aminocarbonylamino, heteroaryloxy,    heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,    —SO-alkyl, —SO-aryl, —SO-heteroaryl, SO₂-aryl and —SO₂-heteroaryl.    Unless otherwise constrained by the definition, all substituents may    optionally be further substituted by 1, 2, or 3 substituents chosen    from alkyl, carboxy, carboxyalkyl, aminocarbonyl, hydroxy, alkoxy,    halogen, CF₃, amino, substituted amino, cyano, and —S(O)_(n)R, where    R is alkyl, aryl, or heteroaryl and n is 0, 1 or 2; or-   (2) an alkylene group as defined above that is interrupted by 1-20    atoms independently chosen from oxygen, sulfur and NR_(a)—, where    R_(a) is chosen from hydrogen, optionally substituted alkyl,    cycloalkyl, cycloalkenyl, aryl, heteroaryl and heterocycyl, or    groups selected from carbonyl, carboxyester, carboxyamide and    sulfonyl; or-   (3) an alkylene group as defined above that has both 1, 2, 3, 4 or 5    substituents as defined above and is also interrupted by 1-20 atoms    as defined above. Examples of substituted alkylenes are    chloromethylene (—CH(Cl)—), aminoethylene (—CH(NH₂)CH₂—),    methylaminoethylene (—CH(NHMe)CH₂—), 2-carboxypropylene isomers    (—CH₂CH(CO₂H)CH₂—), ethoxyethyl (—CH₂CH₂O—CH₂CH₂—),    ethylmethylaminoethyl (—CH₂CH₂N(CH₃)CH₂CH₂—),    1-ethoxy-2-(2-ethoxy-ethoxy)ethane    (—CH₂CH₂O—CH₂CH₂—OCH₂CH₂—OCH₂CH₂—), and the like.

The term “aralkyl” refers to an aryl group covalently linked to analkylene group, where aryl and alkylene are defined herein. “Optionallysubstituted aralkyl” refers to an optionally substituted aryl groupcovalently linked to an optionally substituted alkylene group. Sucharalkyl groups are exemplified by benzyl, phenylethyl,3-(4-methoxyphenyl)propyl, and the like.

The term “alkoxy” refers to the group R—O—, where R is optionallysubstituted alkyl or optionally substituted cycloalkyl, or R is a group—Y—Z, in which Y is optionally substituted alkylene and Z is optionallysubstituted alkenyl, optionally substituted alkynyl; or optionallysubstituted cycloalkenyl, where alkyl, alkenyl, alkynyl, cycloalkyl andcycloalkenyl are as defined herein. Preferred alkoxy groups areoptionally substituted alkyl-O— and include, by way of example, methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy,n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, trifluoromethoxy, and the like.

The term “alkylthio” refers to the group R—S—, where R is as defined foralkoxy.

The term “alkenyl” refers to a monoradical of a branched or unbranchedunsaturated hydrocarbon group preferably having from 2 to 20 carbonatoms, more preferably 2 to 10 carbon atoms and even more preferably 2to 6 carbon atoms and having 1-6, preferably 1, double bond (vinyl).Preferred alkenyl groups include ethenyl or vinyl (—CH═CH₂), 1-propyleneor allyl (—CH₂CH═CH₂), isopropylene (—C(CH₃)═CH₂),bicyclo[2.2.1]heptene, and the like. In the event that alkenyl isattached to nitrogen, the double bond cannot be alpha to the nitrogen.

The term “lower alkenyl” refers to alkenyl as defined above having from2 to 6 carbon atoms.

The term “substituted alkenyl” refers to an alkenyl group as definedabove having 1, 2, 3, 4 or 5 substituents, and preferably 1, 2, or 3substituents, selected from the group consisting of alkyl, alkenyl,alkynyl, alkoxy, cycloalkyl, cycloalkenyl, acyl, acylamino, acyloxy,amino, aminocarbonyl, alkoxycarbonylamino, azido, cyano, halogen,hydroxy, keto, thiocarbonyl, carboxy, carboxyalkyl, arylthio,heteroarylthio, heterocyclylthio, thiol, alkylthio, aryl, aryloxy,heteroaryl, aminosulfonyl, aminocarbonylamino, heteroaryloxy,heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, SO₂-aryl and—SO₂-heteroaryl. Unless otherwise constrained by the definition, allsubstituents may optionally be further substituted by 1, 2, or 3substituents chosen from alkyl, carboxy, carboxyalkyl, aminocarbonyl,hydroxy, alkoxy, halogen, CF₃, amino, substituted amino, cyano, andS(O)_(n)R, where R is alkyl, aryl, or heteroaryl and n is 0, 1 or 2.

The term “alkynyl” refers to a monoradical of an unsaturatedhydrocarbon, preferably having from 2 to 20 carbon atoms, morepreferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbonatoms and having at least 1 and preferably from 1-6-sites of acetylene(triple bond) unsaturation. Preferred alkynyl groups include ethynyl,(—C≡CH), propargyl (or prop-1-yn-3-yl, —CH₂C≡CH), and the like. In theevent that alkynyl is attached to nitrogen, the triple bond cannot bealpha to the nitrogen.

The term “substituted alkynyl” refers to an alkynyl group as definedabove having 1, 2, 3, 4 or 5 substituents, and preferably 1, 2, or 3substituents, selected from the group consisting of alkyl, alkenyl,alkynyl, alkoxy, cycloalkyl, cycloalkenyl, acyl, acylamino, acyloxy,amino, aminocarbonyl, alkoxycarbonylamino, azido, cyano, halogen,hydroxy, keto, thiocarbonyl, carboxy, carboxyalkyl, arylthio,heteroarylthio, heterocyclylthio, thiol, alkylthio, aryl, aryloxy,heteroaryl, aminosulfonyl, aminocarbonylamino, heteroaryloxy,heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, SO₂-aryl and—SO₂-heteroaryl. Unless otherwise constrained by the definition, allsubstituents may optionally be further substituted by 1, 2, or 3substituents chosen from alkyl, carboxy, carboxyalkyl, aminocarbonyl,hydroxy, alkoxy, halogen, CF₃, amino, substituted amino, cyano, and—S(O)_(n)R, where R is alkyl, aryl, or heteroaryl and n is 0, 1 or 2.

The term “aminocarbonyl” refers to the group —C(O)NRR where each R isindependently hydrogen, alkyl, aryl, heteroaryl, heterocyclyl or whereboth R groups are joined to form a heterocyclic group (e.g.,morpholino). Unless otherwise constrained by the definition, allsubstituents may optionally be further substituted by 1-3 substituentschosen from alkyl, carboxy, carboxyalkyl, aminocarbonyl, hydroxy,alkoxy, halogen, CF₃, amino, substituted amino, cyano, and —S(O)_(n)R,where R is alkyl, aryl, or heteroaryl and n is 0, 1 or 2.

The term “acylamino” refers to the group —NRC(O)R where each R isindependently hydrogen, alkyl, aryl, heteroaryl, or heterocyclyl. Unlessotherwise constrained by the definition, all substituents may optionallybe further substituted by 1-3 substituents chosen from alkyl, carboxy,carboxyalkyl, aminocarbonyl, hydroxy, alkoxy, halogen, CF₃, amino,substituted amino, cyano, and —S(O)_(n)R, where R is alkyl, aryl, orheteroaryl and n is 0, 1 or 2.

The term “acyloxy” refers to the groups —O(O)C-alkyl, —O(O)C-cycloalkyl,—O(O)C-aryl, —O(O)C-heteroaryl, and —O(O)C-heterocyclyl. Unlessotherwise constrained by the definition, all substituents may beoptionally further substituted by alkyl, carboxy, carboxyalkyl,aminocarbonyl, hydroxy, alkoxy, halogen, CF₃, amino, substituted amino,cyano, or —S(O)_(n)R, where R is alkyl, aryl, or heteroaryl and n is 0,1 or 2.

The term “aryl” refers to an aromatic carbocyclic group of 6 to 20carbon atoms having a single ring (e.g., phenyl) or multiple rings(e.g., biphenyl), or multiple condensed (fused) rings (e.g., naphthyl oranthryl). Preferred aryls include phenyl, naphthyl and the like.

The term “arylene” refers to a diradical of an aryl group as definedabove. This term is exemplified by groups such as 1,4-phenylene,1,3-phenylene, 1,2-phenylene, 1,4′-biphenylene, and the like.

Unless otherwise constrained by the definition for the aryl or arylenesubstituent, such aryl or arylene groups can optionally be substitutedwith from 1 to 5 substituents, preferably 1 to 3 substituents, selectedfrom the group consisting of alkyl, alkenyl, alkynyl, alkoxy,cycloalkyl, cycloalkenyl, acyl, acylamino, acyloxy, amino,aminocarbonyl, alkoxycarbonylamino, azido, cyano, halogen, hydroxy,keto, thiocarbonyl, carboxy, carboxyalkyl, arylthio, heteroarylthio,heterocyclylthio, thiol, alkylthio, aryl, aryloxy, heteroaryl,aminosulfonyl, aminocarbonylamino, heteroaryloxy, heterocyclyl,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-aryl,—SO-heteroaryl, SO₂-aryl and —SO₂-heteroaryl. Unless otherwiseconstrained by the definition, all substituents may optionally befurther substituted by 1-3 substituents chosen from alkyl, carboxy,carboxyalkyl, aminocarbonyl, hydroxy, alkoxy, halogen, CF₃, amino,substituted amino, cyano, and —S(O)_(n)R, where R is alkyl, aryl, orheteroaryl and n is 0, 1 or 2.

The term “aryloxy” refers to the group aryl-O— wherein the aryl group isas defined above, and includes optionally substituted aryl groups asalso defined above. The term “arylthio” refers to the group R—S—, whereR is as defined for aryl.

The term “amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,cycloalkyl, carboxyalkyl (for example, benzyloxycarbonyl), aryl,heteroaryl and heterocyclyl provided that both R groups are nothydrogen, or a group —Y—Z, in which Y is optionally substituted alkyleneand Z is alkenyl, cycloalkenyl, or alkynyl, Unless otherwise constrainedby the definition, all substituents may optionally be furthersubstituted by 1-3 substituents chosen from alkyl, carboxy,carboxyalkyl, aminocarbonyl, hydroxy, alkoxy, halogen, CF₃, amino,substituted amino, cyano, and —S(O)_(n)R, where R is alkyl, aryl, orheteroaryl and n is 0, 1 or 2.

The term “carboxyalkyl” refers to the groups —C(O)O-alkyl or—C(O)O-cycloalkyl, where alkyl and cycloalkyl, are as defined herein,and may be optionally further substituted by alkyl, alkenyl, alkynyl,alkoxy, halogen, CF₃, amino, substituted amino, cyano, or —S(O)_(n)R, inwhich R is alkyl, aryl, or heteroaryl and n is 0, 1 or 2.

The term “cycloalkyl” refers to carbocyclic groups of from 3 to 20carbon atoms having a single cyclic ring or multiple condensed rings.Such cycloalkyl groups include, by way of example, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, andthe like, or multiple ring structures such as adamantanyl,bicyclo[2.2.1]heptane, 1,3,3-trimethylbicyclo[2.2.1]hept-2-yl,(2,3,3-trimethylbicyclo[2.2.1]hept-2-yl), or carbocyclic groups to whichis fused an aryl group, for example indane, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups having 1,2, 3, 4 or 5 substituents, and preferably 1, 2, or 3 substituents,selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy,cycloalkyl, cycloalkenyl, acyl, acylamino, acyloxy, amino,aminocarbonyl, alkoxycarbonylamino, azido, cyano, halogen, hydroxy,keto, thiocarbonyl, carboxy, carboxyalkyl, arylthio, heteroarylthio,heterocyclylthio, thiol, alkylthio, aryl, aryloxy, heteroaryl,aminosulfonyl, aminocarbonylamino, heteroaryloxy, heterocyclyl,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, SO₂-aryl and —SO₂-heteroaryl. Unlessotherwise constrained by the definition, all substituents may optionallybe further substituted by 1, 2, or 3 substituents chosen from alkyl,carboxy, carboxyalkyl, aminocarbonyl, hydroxy, alkoxy, halogen, CF₃,amino, substituted amino, cyano, and —S(O)_(n)R, where R is alkyl, aryl,or heteroaryl and n is 0, 1 or 2.

The term “halogen” or “halo” refers to fluoro, bromo, chloro, and iodo.

The term “acyl” denotes a group —C(O)R, in which R is hydrogen,optionally substituted alkyl, optionally substituted cycloalkyl,optionally substituted heterocyclyl, optionally substituted aryl, andoptionally substituted heteroaryl.

The term “heteroaryl” refers to a radical derived from an aromaticcyclic group (i.e., fully unsaturated) having 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 carbon atoms and 1, 2, 3 or 4 heteroatomsselected from oxygen, nitrogen and sulfur within at least one ring. Suchheteroaryl groups can have a single ring (e.g., pyridyl or furyl) ormultiple condensed rings (e.g., indolizinyl, benzothiazolyl, orbenzothienyl). Examples of heteroaryls include, but are not limited to,[1,2,4]oxadiazole, [1,3,4]oxadiazole, [1,2,4]thiadiazole,[1,3,4]thiadiazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, isothiazole, phenazine,isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, andthe like as well as N-oxide and N-alkoxy derivatives of nitrogencontaining heteroaryl compounds, for example pyridine-N-oxidederivatives.

The term “heteroarylene” refers to a diradical of a heteroaryl group asdefined above. This term is exemplified by groups such as2,5-imidazolene, 3,5-[1,2,4]oxadiazolene, 2,4-oxazolene, 1,4-pyrazolene,and the like. For example, 1,4-pyrazolene is:

where A represents the point of attachment.

Unless otherwise constrained by the definition for the heteroaryl orheteroarylene substituent, such heteroaryl or heterarylene groups can beoptionally substituted with 1 to 5 substituents, preferably 1 to 3substituents selected from the group consisting of alkyl, alkenyl,alkynyl, alkoxy, cycloalkyl, cycloalkenyl, acyl, acylamino, acyloxy,amino, aminocarbonyl, alkoxycarbonylamino, azido, cyano, halogen,hydroxy, keto, thiocarbonyl, carboxy, carboxyalkyl, arylthio,heteroarylthio, heterocyclylthio, thiol, alkylthio, aryl, aryloxy,heteroaryl, aminosulfonyl, aminocarbonylamino, heteroaryloxy,heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, SO₂-aryl and—SO₂-heteroaryl. Unless otherwise constrained by the definition, allsubstituents may optionally be further substituted by 1-3 substituentschosen from alkyl, carboxy, carboxyalkyl, aminocarbonyl, hydroxy,alkoxy, halogen, CF₃, amino, substituted amino, cyano, and —S(O)_(n)R,where R is alkyl, aryl, or heteroaryl and ti is 0, 1 or 2.

The term “heteroaralkyl” refers to a heteroaryl group covalently linkedto an alkylene group, where heteroaryl and alkylene are defined herein.“Optionally substituted heteroaralkyl” refers to an optionallysubstituted heteroaryl group covalently linked to an optionallysubstituted alkylene group. Such heteroaralkyl groups are exemplified by3-pyridylmethyl, quinolin-8-ylethyl, 4-methoxythiazol-2-ylpropyl, andthe like.

The term “heteroaryloxy” refers to the group heteroaryl-O—.

The term “heterocyclyl” refers to a monoradical saturated or partiallyunsaturated group having a single ring or multiple condensed rings,having from 1 to 40 carbon atoms and from 1 to 10 hetero atoms,preferably 1, 2, 3 or 4 heteroatoms, selected from nitrogen, sulfur,phosphorus, and/or oxygen within the ring. Heterocyclic groups can havea single ring or multiple condensed rings, and includetetrahydrofuranyl, morpholino, piperidinyl, piperazino, dihydropyridino,and the like.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1, 2, 3, 4 or 5, and preferably 1, 2 or 3 substituents, selected fromthe group consisting of alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl,cycloalkenyl, acyl, acylamino, acyloxy, amino, aminocarbonyl,alkoxycarbonylamino, azido, cyano, halogen, hydroxy, keto, thiocarbonyl,carboxy, carboxyalkyl, arylthio, heteroarylthio, heterocyclylthio,thiol, alkylthio, aryl, aryloxy, heteroaryl, aminosulfonyl,aminocarbonylamino, heteroaryloxy, heterocyclyl, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl,SO₂ alkyl, SO₂-aryl and —SO₂-heteroaryl. Unless otherwise constrained bythe definition, all substituents may optionally be further substitutedby 1-3 substituents chosen from alkyl, carboxy, carboxyalkyl,aminocarbonyl, hydroxy, alkoxy, halogen, CF₃, amino, substituted amino,cyano, and —S(O)_(n)R, where R is alkyl, aryl, or heteroaryl and n is 0,1 or 2.

The term “thiol” refers to the group —SH.

The term “substituted alkylthio” refers to the group —S-substitutedalkyl.

The term “heteroarylthiol” refers to the group —S-heteroaryl wherein theheteroaryl group is as defined above including optionally substitutedheteroaryl groups as also defined above.

The term “sulfoxide” refers to a group —S(O)R, in which R is alkyl,aryl, or heteroaryl. “Substituted sulfoxide” refers to a group —S(O)R,in which R is substituted alkyl, substituted aryl, or substitutedheteroaryl, as defined herein.

The term “sulfone” refers to a group —S(O)₂R, in which R is alkyl, aryl,or heteroaryl. “Substituted sulfone” refers to a group —S(O)₂R, in whichR is substituted alkyl, substituted aryl, or substituted heteroaryl, asdefined herein.

The term “keto” refers to a group —C(O)—.

The term “thiocarbonyl” refers to a group —C(S)—.

The term “carboxy” refers to a group —C(O)—OH.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not.

The term “compound of Formula I and Formula II” is intended to encompassthe compounds of the invention as disclosed, and the pharmaceuticallyacceptable salts, pharmaceutically acceptable esters, prodrugs, hydratesand polymorphs of such compounds. Additionally, the compounds of theinvention may possess one or more asymmetric centers, and can beproduced as a racemic mixture or as individual enantiomers ordiastereoisomers. The number of stereoisomers present in any givencompound of Formula I depends upon the number of asymmetric centerspresent (there are 2^(n) stereoisomers possible where n is the number ofasymmetric centers). The individual stereoisomers may be obtained byresolving a racemic or non-racemic mixture of an intermediate at someappropriate stage of the synthesis, or by resolution of the compound ofFormula I by conventional means. The individual stereoisomers (includingindividual enantiomers and diastereoisomers) as well as racemic andnon-racemic mixtures of stereoisomers are encompassed within the scopeof the present invention, all of which are intended to be depicted bythe structures of this specification unless otherwise specificallyindicated.

“Isomers” are different compounds that have the same molecular formula.

“Stereoisomers” are isomers that differ only in the way the atoms arearranged in space.

“Enantiomers” are a pair of stereoisomers that are non-superimposablemirror images of each other. A 1:1 mixture of a pair of enantiomers is a“racemic” mixture. The term “(±)” is used to designate a racemic mixturewhere appropriate.

“Diastereoisomers” are stereoisomers that have at least two asymmetricatoms, but which are not mirror-images of each other.

The absolute stereochemistry is specified according to theCahn-Ingold-Prelog R—S system. When the compound is a pure enantiomerthe stereochemistry at each chiral carbon may be specified by either Ror S. Resolved compounds whose absolute configuration is unknown aredesignated (+) or (−) depending on the direction (dextro- orlaevorotary) which they rotate the plane of polarized light at thewavelength of the sodium D line.

“Topical administration” shall be defined as the delivery of thetherapeutic agent to the surface of the wound and adjacent epithelium.

“Parenteral administration” is the systemic delivery of the therapeuticagent via injection to the patient.

The term “therapeutically effective amount” refers to that amount of acompound of Formula I that is sufficient to effect treatment, as definedbelow, when administered to a mammal in need of such treatment. Thetherapeutically effective amount will vary depending upon the specificactivity of the therapeutic agent being used, and the age, physicalcondition, existence of other disease states, and nutritional status ofthe patient. Additionally, other medication the patient may be receivingwill effect the determination of the therapeutically effective amount ofthe therapeutic agent to administer.

The term “treatment” or “treating” means any treatment of a disease in amammal, including:

-   (i) preventing the disease, that is, causing the clinical symptoms    of the disease not to develop;-   (ii) inhibiting the disease, that is, arresting the development of    clinical symptoms; and/or-   (iii) relieving the disease, that is, causing the regression of    clinical symptoms.

In many cases, the compounds of this invention are capable of formingacid and/or base salts by virtue of the presence of amino and/orcarboxyl groups or groups similar thereto. The term “pharmaceuticallyacceptable salt” refers to salts that retain the biologicaleffectiveness and properties of the compounds of Formula I, and whichare not biologically or otherwise undesirable. Pharmaceuticallyacceptable base addition salts can be prepared from inorganic andorganic bases. Salts derived from inorganic bases, include by way ofexample only, sodium, potassium, lithium, ammonium, calcium andmagnesium salts. Salts derived from organic bases include, but are notlimited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl)amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenylamities, di(substituted alkenyl)amines, tri(substituted alkenyl)amines,cycloalkyl amines, di(cycloalkyl)amines, tri(cycloalkyl)amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl)amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group.

Specific examples of suitable amines include, by way of example only,isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine,tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine,purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and thelike.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

Nomenclature

The naming and numbering of the compounds of the invention isillustrated with a representative compound of Formula I in which R¹ isn-propyl, R² and R³ are hydrogen, X is pyrazol-4-ylene, Y is methylene,and Z is 5-(4-chlorophenyl)(1,2,4-oxadiazol-3-yl,

which is named:

-   8-(1-{[5-(4-chlorophenyl)(1,2,4-oxadiazol-3-yl)]methyl}pyrazol-4-yl)-1-propyl-1,3,7-trihydropurine-2,6-dione.    The Method of the Invention

The present invention relates to methods of preventing and/or treatinghepatic disease by administration of a therapeutically effective amountof a A_(2B) adenosine receptor antagonist to a mammal in need thereof.While not wishing to be bound by theory, it is believed that the abilityA_(2B) adenosine receptor antagonist to prevent fibrogenesis providesthese compounds with the ability to prevent and treat hepatic disease.

As hepatic fibrogenesis is a significant component of viral andalcoholic hepatitis, Wilson's disease, hemochromatosis, steatosis, andnonalcoholic steatohepatitis, and may be the result of a surgicalinsult, i.e, liver replacement or repair, or hepatotoxic medicaltreatment, i.e., radiation, chemotherapy drugs, antibiotics,antiemetics, and the like, the method of the invention will generallyinvolve administration of an A_(2B) adenosine receptor antagonist to apatient suffering from one of the aforementioned conditions orundergoing hepatotoxic treatment.

Chemotherapy and radiation therapy in the treatment of cancer are two ofthe most common types of hepatotoxic treatment. Hepatotoxic drugs usedto treat cancer include, but are not limited to, adriamycin,methotrexate, 6 mercaptopurine, carboplatin, DTIC (dacarbazine), BiCNU,L-asparaginase, and pentostatin.

Other drugs known to have hepatotoxic side effects include, but are notlimited to, acebutolol; acetaminophen; actinomycin d; adrenocorticalsteroids; adriamycin; allopurinol; amoxicillin/clavulanate; anabolicsteroids; anti-inflammatory drugs; antithyroid drugs; aspirin; atenolol;azathioprine; captopril; carbamazepine; carbimazole; carmustine;cephalosporins; chlordiazepoxide; chlorpromazine;chlorpromazine/valproic acid; chlorpropamide;chlorpropamide/erythromycin (combination); cimetidine; cloxacillinflecainide; cyclophosphamide; cyclophosphamide/cyclosporine;cyclosporine; dacarbazine; danazol; dantrolene; diazepam; diclofenac;diltiazem; disopyramide; enalapril; enflurane; erythromycin; ethambutol;ethionamide; flurazepam; flutamide; glyburide; gold; griseofulvin;haloperidol; halothane; hydralazine; ibuprofen; imipramine;indomethacin; isoniazid; ketoconazole; labetalol; maprotiline;mercaptopurine; methotrexate; methyldopa; methyltestosterone;metoprolol; mianserin; mitomycin; naproxen; nicotinic acid; nifedipine;nitrofurantoin; nonsteroidal; norethandrolone; oral contraceptives;oxacillin; para-aminosalicylic acid; penicillamine; penicillin;penicillins; phenelzine; phenindione; phenobarbital; phenothiazines;phenylbutazone; phenyloin; phenyloin troleandomycin; piroxicam;probenecid; procainamide; propoxyphene; pyrazinamide; quinidine;quinine; ranitidine; salicylates; sulfonamides; sulindac; tamoxifen;terbinafine HCl (Lamisil, Sporanox); testosterone; tetracyclines;thiabendazole; thioquanine; thorotrast; tolbutamide; tricyclicantidepressants; valproic acid; verapamil; vincristine; and vitamin A.

The onset of hepatic fibrogenesis begins with activation of humanhepatic stellate cells (HHSC). Upon activation, HHSCs begin tosynthesize a fibrotic matrix that is rich in type I collagen. Thisfibrotic matrix ultimately results in the scarring traditionallyreferred to as hepatic fibrosis. Applicants have surprisingly discoveredthat the A_(2B) adenosine receptor is involved in the development ofhepatic fibrosis as activation of the A_(2B) receptor in HHSCs inducesthe release of IL-6 and expression of a-smooth muscle actin, a markerfor HHSC activation. The release of IL-6 has in turn been shown tostimulate collagen production. It has been discovered that inhibition ofthe A_(2B) receptor reduces the NECA-induced IL-6 release and collagenproduction.

The A_(2B) adenosine receptor antagonist is administered systemically aseither an oral or IV formulation but may also be administered directlyto the hepatic tissue via injection. This administration can be as asingle dose or as repeated doses given at multiple designated intervals.It will readily be appreciated by those skilled in the art that thepreferred dosage regimen will vary with the patient and severity of thecondition being treated.

Pharmaceutical Compositions

When selected as the adenosine A_(2B) receptor antagonist, the compoundsof Formula I are usually administered in the form of pharmaceuticalcompositions. This invention therefore provides pharmaceuticalcompositions that contain, as the active ingredient, one or more of thecompounds of Formula I or Formula II, or a pharmaceutically acceptablesalt or ester thereof, and one or more pharmaceutically acceptableexcipients, carriers, including inert solid diluents and fillers,diluents, including sterile aqueous solution and various organicsolvents, solubilizers and adjuvants. The compounds of Formula I and/orFormula II may be administered alone or in combination with othertherapeutic agents. Such compositions are prepared in a manner wellknown in the pharmaceutical art (see, e.g., Remington's PharmaceuticalSciences, Mace Publishing Co., Philadelphia, Pa. 17^(th) Ed. (1985) and“Modern Pharmaceutics”, Marcel Dekker, Inc. 3^(rd) Ed. (G. S. Banker &C. T. Rhodes, Eds.).

The A_(2B) Adenosine Receptor Antagonists

Any A_(2B) adenosine receptor antagonist may be used in the method ofthe invention. Numerous compounds that antagonize the A_(2B) receptorare known in the art, as are methods for determining if a specificcompound has such activity. For example, a review article by Feoktistovand Baggioni, ((1997) Pharmacological Reviews 49:381-402) reports thebinding affinity of eight adenosine receptor agonists and eightantagonists for all four subtypes of adenosine receptors. Referencescited therein provide detailed descriptions of the procedures used.(Robeva et al, (1996) J. Drug Dev. Res 39:243-252; Jacobson et al (1996)Drug Dev. Res. 39:289-300; Feoktistov and Baggioni (1993) MolecularPharmacology 43:909-914). Effective methods for determining the bindingaffinity of a compound for a receptor use a radiolabeled agonist orantagonist and correlation of the binding of that compound to a membranefraction known to contain that receptor; for example, to determinewhether a compound is an A_(2B) antagonist, the membrane fraction wouldcontain the A_(2B) adenosine receptor. Another particularly effectiveprocedure for determining whether a compound is an A_(2B) antagonist isreported in U.S. Pat. No. 5,854,081.

Compounds selective for the A_(2B) receptor subtype are thereforepreferred for the present methods. An example, but not a limitation, ofsuch a compound is 3-n-propylxanthine (enprofylline). Suitable compoundsare also disclosed in U.S. Pat. No. 6,545,002. Compounds that antagonizeother receptors in addition to the A_(2B) receptor are also suitable foruse in the present invention. One example of such a compound is1,3-dipropyl-8-(p-acrylic)phenylxanthine.

One particularly preferred class of A_(2B) adenosine receptorantagonists are those disclosed in copending and commonly assigned U.S.Pat. No. 6,825,349, in copending and commonly assigned U.S. patentapplication Ser. No. 10/719,102, which published as U.S. PatentApplication Publication No. 20040176399, and in copending and commonlyassigned U.S. patent application Ser. No. 11/453,414, which published asU.S. Patent Application Publication No. 20060293283. The compoundsdisclosed in these applications have the structure of Formula I, II, andIII as presented in the Summary of the Invention above and can besynthesized as described in the references or as detailed below.

Synthetic Reaction Parameters

The terms “solvent”, “inert organic solvent” or “inert solvent” mean asolvent inert under the conditions of the reaction being described inconjunction therewith [including, for example, benzene, toluene,acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”),chloroform, methylene chloride (or dichloromethane), diethyl ether,methanol, pyridine and the like]. Unless specified to the contrary, thesolvents used in the reactions of the present invention are inertorganic solvents, and the reactions are carried out under an inert gas,preferably nitrogen.

The term “q.s.” means adding a quantity sufficient to achieve a statedfunction, e.g., to bring a solution to the desired volume (i.e., 100%).

Synthesis of the Compounds of Formula I and II

One preferred method of preparing compounds of Formula I or II where R³is hydrogen is shown in Reaction Scheme I.

Step 1—Preparation of Formula (2)

The compound of formula (2) is made from the compound of formula (1) bya reduction step. Conventional reducing techniques may be used, forexample using sodium dithionite in aqueous ammonia solution; preferably,reduction is carried out with hydrogen and a metal catalyst. Thereaction is carried out at in an inert solvent, for example methanol, inthe presence of a catalyst, for example 10% palladium on carboncatalyst, under an atmosphere of hydrogen, preferably under pressure,for example at about 30 psi, for about 2 hours. When the reaction issubstantially complete, the product of formula (2) is isolated byconventional means to provide a compound of formula (2).

Step 2—Preparation of Formula (3)

The compound of formula (2) is then reacted with a carboxylic acid ofthe formula Z—Y—X—CO₂H in the presence of a carbodiimide, for example1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. Thereaction is conducted in a protic solvent, for example methanol,ethanol, propanol, and the like, preferably methanol, at a temperatureof about 20-30° C., preferably about room temperature, for about 12-48hours, preferably about 16 hours. When the reaction is substantiallycomplete, the product of formula (3) is isolated conventionally, forexample by removal of the solvent under reduced pressure, and washingthe product. Alternatively, the next step can be carried out without anyfurther purification.

Alternative Preparation of a Compound of Formula (3)

Alternatively, the carboxylic acid of the formula Z—Y—X—CO₂H is firstconverted to an acid halide of the formula Z—Y—X—C(O)L, where L ischloro or bromo, by reacting with a halogenating agent, for examplethionyl chloride or thionyl bromide, preferably thiony chloride.Alternatively, oxalyl chloride, phosphorus pentachloride or phosphorusoxychloride may be used. The reaction is preferably conducted in theabsence of a solvent, using excess halogenating agent, for example at atemperature of about 60-80° C., preferably about 70° C., for about 1-8hours, preferably about 4 hours. When the reaction is substantiallycomplete, the product of formula Z—Y—X—C(O)L is isolated conventionally,for example by removal of the excess halogenating agent under reducedpressure.

The product is then reacted with a compound of formula (2) in an inertsolvent, for example acetonitrile, in the presence of a tertiary base,for example triethylamine. The reaction is conducted at an initialtemperature of about 0 C, and then allowed to warm to 20-30° C.,preferably about room temperature, for about 12-48 hours, preferablyabout 16 hours. When the reaction is substantially complete, the productof formula (3) is isolated conventionally, for example by diluting thereaction mixture with water, filtering off the product, and washing theproduct with water followed by ether.

Step 3—Preparation of Formula I

The compound of formula (3) is then converted into a compound of FormulaI by a cyclization reaction. The reaction is conducted in a proticsolvent, for example methanol, ethanol, propanol, and the like,preferably methanol, in the presence of a base, for example potassiumhydroxide, sodium hydroxide, sodium methoxide, sodium ethoxide,potassium t-butoxide, preferably aqueous sodium hydroxide, at atemperature of about 50-80° C., preferably about 80° C., for about 1-8hours, preferably about 3 hours. When the reaction is substantiallycomplete, the product of Formula I is isolated conventionally, forexample by removal of the solvent under reduced pressure, acidifying theresidue with an aqueous acid, filtering off the product, then washingand drying the product.

The compound of formula (1) may be prepared by various methods. Onepreferred method is shown in Reaction Scheme II.

Step 1—Preparation of Formula (5)

The compound of formula (4) is either commercially available or preparedby means well known in the art. It is reacted with ethyl cyanoacetate ina protic solvent, for example ethanol, in the presence of a strong base,for example sodium ethoxide. The reaction is carried out at about refluxtemperature, for about 4 to about 24 hours. When the reaction issubstantially complete, the compound of formula (5) thus produced isisolated conventionally.

Step 2 and 3—Preparation of Formula (7)

The compound of formula (5) is reacted with the dimethylacetal ofN,N-dimethylformamide in a polar solvent, for exampleN,N-dimethylformamide. The reaction is carried out at about 40° C., forabout 1 hour. When the reaction is substantially complete, the compoundof formula (6) thus produced is reacted with a compound of formulaR¹Hal, where Hal is chloro, bromo, or iodo, in the presence of a base,for example potassium carbonate. The reaction is carried out at about80° C., for about 4-24 hour. When the reaction is substantiallycomplete, the product of formula (7) is isolated conventionally, forexample by evaporation of the solvents under reduced pressure, and theresidue is used in the next reaction with no further purification.

Step 4—Preparation of Formula (8)

The compound of formula (7) is reacted with aqueous ammonia in a polarsolvent, for example suspended in methanol. The reaction is carried outat about room temperature, for about 1-3 days. When the reaction issubstantially complete, the product of formula (8) is isolatedconventionally, for example by chromatography over a silica gel column,eluting, for example, with a mixture of dichloromethane/methanol.

Step 5—Preparation of Formula (1)

The compound of formula (8) is then mixed with sodium nitrite in anaqueous acidic solvent, preferably acetic acid and water, for example50% acetic acid/water. The reaction is carried out at a temperature ofabout 50-90° C., preferably about 70° C., for about 1 hour. When thereaction is substantially complete, the product of formula (1) isisolated by conventional means.

Alternatively, the reaction may be conducted in an aqueous solvent, forexample dimethylformamide and water, and reacted with a strong acid, forexample hydrochloric acid.

A compound of formula (8) can be prepared from a compound of formula(10) using a similar method, as shown in Reaction Scheme IIA.

Step 2 and 3—Preparation of Formula (7)

The compound of formula (10) is reacted with the dimethylacetal ofN,N-dimethylformamide in a polar solvent, for exampleN,N-dimethylformamide. The reaction is carried out at about 40° C., forabout 1 hour. When the reaction is substantially complete, the compoundof formula (6a) thus produced is reacted with a compound of formulaR²Hal, where Hal is chloro, bromo, or iodo, in the presence of a base,for example potassium carbonate. The reaction is carried out at about80° C., for about 4-24 hour. When the reaction is substantiallycomplete, the product of formula (7) is isolated conventionally, forexample by evaporation of the solvents under reduced pressure, and theresidue is used in the next reaction with no further purification.

Step 4—Preparation of Formula (8)

The compound of formula (7) is reacted with aqueous ammonia in a polarsolvent, for example suspended in methanol. The reaction is carried outat about room temperature, for about 1-3 days. When the reaction issubstantially complete, the product of formula (8) is isolatedconventionally, for example by chromatography over a silica gel column,eluting, for example, with a mixture of dichloromethane/methanol.

The compound of formula (3) may also be prepared by various methods. Onepreferred method is shown in Reaction Scheme III.

Step 1—Preparation of Formula (10)

The commercially available compound 6-aminouracil is first silylated,for example by reaction with excess hexamethyldisilazane as a solvent inthe presence of a catalyst, for example ammonium sulfate. The reactionis carried out at about reflux temperature, for about 1-10 hours. Whenthe reaction is substantially complete, the silylated compound thusproduced is isolated conventionally, and then reacted with a compound offormula R¹Hal, where Hal is chloro, bromo, or iodo, preferably in theabsence of a solvent. The reaction is carried out at about reflux, forabout 4-48 hours, preferably about 12-16 hours. When the reaction issubstantially complete, the product of formula (10) is isolated byconventional means.

Step 2—Preparation of Formula (11)

The compound of formula (10) is then dissolved in an aqueous acid, forexample aqueous acetic acid, and reacted with sodium nitrite. Thereaction is carried out at a temperature of about 20-50° C., preferablyabout 30° C., over about 30 minutes. When the reaction is substantiallycomplete, the product of formula (11) is isolated by conventional means,for example by filtration.

Step 3—Preparation of Formula (12)

The compound of formula (11) is then reduced to a diamino derivative. Ingeneral, the compound of formula (11) is dissolved in aqueous ammonia,and then a reducing agent, for example sodium hydrosulfite, added. Thereaction is conducted at a temperature of about 70° C. When the reactionis substantially complete, the product of formula (12) is isolatedconventionally, for example by filtration of the cooled reactionmixture.

Step 4—Preparation of Formula (13)

The compound of formula (12) is then reacted with a carboxylic acid ofthe formula Z—Y—X—CO₂H in the presence of a carbodiimide, for example1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. Thereaction is conducted at a temperature of about 20-30° C., for about12-48 hours. When the reaction is substantially complete, the product offormula (13) is isolated conventionally, for example by filtration ofthe cooled reaction mixture.

Alternatively, the carboxylic acid of the formula Z—Y—X—CO₂H isconverted to an acid halide of the formula Z—Y—X—C(O)L, where L ischloro or bromo, by reacting with a halogenating agent, for examplethionyl chloride or thionyl bromide; alternatively, phosphoruspentachloride or phosphorus oxychloride may be used. The reaction ispreferably conducted in the absence of a solvent, using excesshalogenating agent, for example at a temperature of about 60-80° C.,preferably about 70° C., for about 1-8 hours, preferably about 4 hours.When the reaction is substantially complete, the product of formulaZ—Y—X—C(O)L is isolated conventionally, for example by removal of theexcess halogenating agent under reduced pressure.

The product of the formula Z—Y—X—C(O)L is then reacted with a compoundof formula (12) in an inert solvent, for example acetonitrile, in thepresence of a tertiary base, for example triethylamine. The reaction isconducted at an initial temperature of about 0 C, and then allowed towarm to 20-30° C., preferably about room temperature, for about 12-48hours, preferably about 16 hours. When the reaction is substantiallycomplete, the product of formula (13) is isolated conventionally, forexample by diluting the reaction mixture with water, filtering off theproduct, and washing the product with water followed by ether.

Step 5—Preparation of Formula (3)

The compound of formula (13) is reacted with a compound of formulaR²Hal, where Hal is chloro, bromo, or iodo, in the presence of a base,for example potassium carbonate. The reaction is carried out at aboutroom temperature, for about 4-24 hour, preferably about 16 hours. Whenthe reaction is substantially complete, the product of formula (3) isisolated conventionally, for example by evaporation of the solventsunder reduced pressure, and the residue may be purified conventionally,or may be used in the next reaction with no further purification.

Another method of preparing a compound of formula (3) is shown inReaction Scheme IV.

Step 1—Preparation of Formula (14)

The compound of formula (5) is then mixed with sodium nitrite in anaqueous acidic solvent, preferably acetic acid and water, for example50% acetic acid/water. The reaction is carried out at a temperature ofabout 50-90° C., preferably about 70° C., for about 1 hour. When thereaction is substantially complete, the product of formula (14) isisolated by conventional means.

Alternatively, the reaction may be conducted in an aqueous solvent, forexample dimethylformamide and water, and reacted with a strong acid, forexample hydrochloric acid.

Step 3—Preparation of Formula (15)

The compound of formula (14) is then reduced to a diamino derivative. Ingeneral, the compound of formula (14) is dissolved in aqueous ammonia,and then a reducing agent, for example sodium hydrosulfite, added. Thereaction is conducted at a temperature of about 70° C. When the reactionis substantially complete, the product of formula (15) is isolatedconventionally, for example by filtration of the cooled reactionmixture.

Step 4—Preparation of Formula (16)

The compound of formula (15) is then reacted with a carboxylic acid ofthe formula Z—Y—X—CO₂H in the presence of a carbodiimide, for example1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. Thereaction is conducted at a temperature of about 20-30° C., for about12-48 hours, in an inert solvent, for example methanol. When thereaction is substantially complete, the product of formula (16) isisolated conventionally, for example by filtration of the cooledreaction mixture.

Alternatively, the carboxylic acid of the formula Z—Y—X—CO₂H isconverted to an acid halide of the formula Z—Y—X—C(O)L, where L ischloro or bromo, by reacting with a halogenating agent, for examplethionyl chloride or thionyl bromide; alternatively, phosphoruspentachloride or phosphorus oxychloride may be used. The reaction ispreferably conducted in the absence of a solvent, using excesshalogenating agent, for example at a temperature of about 60-80° C.,preferably about 70° C., for about 1-8 hours, preferably about 4 hours.When the reaction is substantially complete, the product of formulaZ—Y—X—C(O)L is isolated conventionally, for example by removal of theexcess halogenating agent under reduced pressure.

The product of the formula Z—Y—X—C(O)L is then reacted with a compoundof formula (15) in an inert solvent, for example acetonitrile, in thepresence of a tertiary base, for example triethylamine. The reaction isconducted at an initial temperature of about 0 C, and then allowed towarm to 20-30° C., preferably about room temperature, for about 12-48hours, preferably about 16 hours. When the reaction is substantiallycomplete, the product of formula (16) is isolated conventionally, forexample by diluting the reaction mixture with water, filtering off theproduct, and washing the product with water followed by ether.

Step 5—Preparation of Formula (3)

The compound of formula (16) is reacted with a compound of formulaR¹Hal, where Hal is chloro, bromo, or iodo, in the presence of a base,for example potassium carbonate. The reaction is carried out at about80° C., for about 4-24 hour, preferably about 16 hours. When thereaction is substantially complete, the product of formula (3) isisolated conventionally, for example by evaporation of the solventsunder reduced pressure, and the residue may be purified conventionally,or may be used in the next reaction with no further purification.

An example of a synthesis of a compound of Z—Y—X—CO₂H in which X ispyrazol-1,4-yl, Y is methylene, and Z is 3-trifluoromethylphenyl, isshown in Reaction Scheme V.

Ethyl pyrazole-4-carboxylate is reacted with1-(bromomethyl)-3-(trifluoromethyl)benzene in acetone in the presence ofpotassium carbonate. The product, ethyl1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylate, is thenhydrolyzed with potassium hydroxide in methanol, to provide1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylic acid.

Utility Testing and Administration

General Utility

The method and pharmaceutical compositions of the invention areeffective in the prevention and treatment of hepatic disease such ashepatic fibrosis and/or hepatic inflammation in a mammal. Typical causesof hepatic disease include, but are not limited to, viral and alcoholichepatitis, Wilson's disease, hemochromatosis, steatosis, andnonalcoholic steatohepatitis (NASH). Hepatic disease may also result asa consequence of surgical intervention, i.e., liver replacement orrepair, or as a consequence of drug-induced liver damage.

Testing

Activity testing is conducted as described in those patents and patentapplications referenced above, and in the Examples below, and by methodsapparent to one skilled in the art.

Administration

The compounds of Formula I may be administered in either single ormultiple doses by any of the accepted modes of administration of agentshaving similar utilities, for example as described in those patents andpatent applications incorporated by reference, including buccal,intranasal, intra-arterial injection, intravenously, intraperitoneally,parenterally, intramuscularly, subcutaneously, orally, or as aninhalant.

Oral administration is the preferred route for administration of thecompounds of Formula I. Administration may be via capsule or entericcoated tablets, or the like. In making the pharmaceutical compositionsthat include at least one compound of Formula I, the active ingredientis usually diluted by an excipient and/or enclosed within such a carrierthat can be in the form of a capsule, sachet, paper or other container.When the excipient serves as a diluent, in can be a solid, semi-solid,or liquid material (as above), which acts as a vehicle, carrier ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, syrups, aerosols (as a solid or in aliquid medium), ointments containing, for example, up to 10% by weightof the active compound, soft and hard gelatin capsules, sterileinjectable solutions, and sterile packaged powders.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents.

The compositions of the invention can be formulated so as to providequick, sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.Controlled release drug delivery systems for oral administration includeosmotic pump systems and dissolutional systems containing polymer-coatedreservoirs or drug-polymer matrix formulations. Examples of controlledrelease systems are given in U.S. Pat. Nos. 3,845,770; 4,326,525;4,902,514; and 5,616,345. Another formulation for use in the methods ofthe present invention employs transdermal delivery devices (“patches”).Such transdermal patches may be used to provide continuous ordiscontinuous infusion of the compounds of the present invention incontrolled amounts. The construction and use of transdermal patches forthe delivery of pharmaceutical agents is well known in the art. See,e.g., U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patchesmay be constructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents.

Adenosine A_(2B) receptor antagonists such as the compounds of Formula Iare effective over a wide dosage range and is generally administered ina pharmaceutically effective amount. Typically, for oral administration,each dosage unit contains from 1 mg to 2 g of an adenosine A_(2B)receptor antagonist, more commonly from 1 to 700 mg, and for parenteraladministration, from 1 to 700 mg of an adenosine A_(2B) receptorantagonist, more commonly about 2 to 200 mg. It will be understood,however, that the amount of the adenosine A_(2B) receptor antagonistactually administered will be determined by a physician, in the light ofthe relevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered and itsrelative activity, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction, or to protect from the acid conditions of the stomach. Forexample, the tablet or pill can comprise an inner dosage and an outerdosage component, the latter being in the form of an envelope over theformer. The two components can be separated by an enteric layer thatserves to resist disintegration in the stomach and permit the innercomponent to pass intact into the duodenum or to be delayed in release.A variety of materials can be used for such enteric layers or coatings,such materials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol, andcellulose acetate.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. Preferably the compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be inhaled directly from thenebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices that deliver the formulationin an appropriate manner.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Preparation of a Compound of Formula (5) A. Preparation of aCompound of Formula (5) in which R² is Ethyl

A solution of sodium ethoxide was prepared from sodium (4.8 g, 226 mmol)and dry ethanol (150 ml). To this solution was added amino-N-ethylamide(10 g, 113 m mol) and ethyl cyanoacetate (12.8 g, 113 mmol). Thisreaction mixture was stirred at reflux for 6 hours, cooled, and solventremoved from the reaction mixture under reduced pressure. The residuewas dissolved in water (50 ml), and the pH adjusted to 7 withhydrochloric acid. The mixture was allowed to stand overnight at 0° C.,and the precipitate filtered off, washed with water and air-dried, toprovide 6-amino-1-ethyl-1,3-dihydropyrimidine-2,4-dione, a compound offormula (5).

¹H-NMR (DMSO-d6) δ 10.29 (s, 1H), 6.79 (s, 2H), 4.51 (s, 1H), 3.74-3.79(m, 2H), 1.07 (t, 3H, J=7.03 Hz); MS m/z 155.98 (M⁺), 177.99 (M⁺+Na)

B. Preparation of a Compound of Formula (5) in which R² is Methyl

Similarly, following the procedure of Example 1A, but replacingamino-N-ethylamide with amino-N-methylamide,6-amino-1-methyl-1,3-dihydropyrimidine-2,4-dione was prepared.

C. Preparation of a Compound of Formula (5) Varying R²

Similarly, following the procedure of Example 1A, but replacingamino-N-ethylamide with other compounds of formula (4), other compoundsof formula (5) are prepared.

Example 2 A. Preparation of a Compound of Formula (6) Preparation of aCompound of Formula (6) in which R² is Ethyl

A suspension of 6-amino-1-ethyl-1,3-dihydropyrimidine-2,4-dione (0.77 g,5 mmol) in anhydrous N,N-dimethylacetamide (25 ml) andN,N-dimethylformamide dimethylacetal (2.7 ml, 20 mmol) and was warmed at40° C. for 90 minutes. Solvent was then removed under reduced pressure,and the residue triturated with ethanol, filtered, and washed withethanol, to provide6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-1,3-dihydropyrimidine-2,4-dione,a compound of formula (6).

¹H-NMR (DMSO-d6) δ 10.62 (s, 1H), 8.08 (s, 1H), 4.99 (s, 1H), 3.88-3.95(m, 2H), 3.13 (s, 3H), 2.99 (s, 3H), 1.07 (t, 3H, J=7.03 Hz); MS m/z210.86 (M⁺), 232.87 (M⁺+Na)

B. Preparation of a Compound of Formula (6) in which R² is Methyl

Similarly, following the procedure of Example 2A, but replacing6-amino-1-ethyl-1,3-dihydropyrimidine-2,4-dione with6-amino-1-methyl-1,3-dihydropyrimidine-2,4-dione,6-[2-(dimethylamino)-1-azavinyl]-1-methyl-1,3-dihydropyrimidine-2,4-dionewas prepared.

C. Preparation of a Compound of Formula (6) Varying R²

Similarly, following the procedure of Example 2A, but replacing6-amino-1-ethyl-1,3-dihydropyrimidine-2,4-dione with other compounds offormula (5), other compounds of formula (6) are prepared.

Example 3 Preparation of a Compound of Formula (7) A. Preparation of aCompound of Formula (7) in which R¹ is n-Propyl and R² is Ethyl

A mixture of a solution of6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-1,3-dihydropyrimidine-2,4-dione(1.5 g, 7.1 mmol) in dimethylformamide (25 ml), potassium carbonate (1.5g, 11 mmol) and n-propyl iodide (1.54 g, 11 mmol) was stirred at 80° C.for 5 hours. The reaction mixture was cooled to room temperature,filtered, the solvents were evaporated and the product of formula (7),6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dione,was used as such in the next reaction.

B. Preparation of a Compound of Formula (7), Varying R¹ and R²

Similarly, following the procedure of Example 3A, but replacing6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-1,3-dihydropyrimidine-2,4-dionewith other compounds of formula (6), the following compounds of formula(7) were prepared:

-   6-[2-(dimethylamino)-1-azavinyl]-1-methyl-3-propyl-1,3-dihydropyrimidine-2,4-dione.-   6-[2-(dimethylamino)-1-azavinyl]-1-methyl-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4-dione;-   6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4-dione;-   6-[2-(dimethylamino)-1-azavinyl]-1-methyl-3-(2-methylpropyl)-1,3-dihydropyrimidine-2,4-dione;    and-   6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-3-(2-methylpropyl)-1,3-dihydropyrimidine-2,4-dione.

C. Preparation of a Compound of Formula (7), Varying R¹ and R²

Similarly, following the procedure of Example 3A, but replacing6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-1,3-dihydropyrimidine-2,4-dionewith other compounds of formula (6), other compounds of formula (7) areprepared.

Example 4 Preparation of a Compound of Formula (8) A. Preparation of aCompound of Formula (8) in which R¹ is n-Propyl and R² is Ethyl

A solution of6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dione(2.1 g) was dissolved in a mixture of methanol (10 ml) and 28% aqueousammonia solution (20 ml), and stirred for 72 hours at room temperature.Solvent was then removed under reduced pressure, and the residuepurified by chromatography on a silica gel column, eluting with amixture of dichloromethane/methanol (15/1), to provide6-amino-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dione, a compound offormula (8).

¹H-NMR (DMSO-d6) δ 6.80 (s, 2H), 4.64 (s, 1H), 3.79-3.84 (m, 2H),3.63-3.67 (m, 2H), 1.41-1.51 (m, 2H), 1.09 (t, 3H, J=7.03 Hz), 0.80 (t,3H, J=7.42 Hz); MS m/z 197.82 (M⁺)

B. Preparation of a Compound of Formula (8), Varying R¹ and R²

Similarly, following the procedure of Example 4A, but replacing6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dionewith other compounds of formula (7), the following compounds of formula(8) were prepared:

-   6-amino-1-methyl-3-propyl-1,3-dihydropyrimidine-2,4-dione;-   6-amino-1-methyl-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4-dione;-   6-amino-1-ethyl-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4-dione;-   6-amino-1-methyl-3-(2-methylpropyl)-1,3-dihydropyrimidine-2,4-dione;    and-   6-amino-1-ethyl-3-(2-methylpropyl)-1,3-dihydropyrimidine-2,4-dione.

C. Preparation of a Compound of Formula (7) Varying R¹ and R²

Similarly, following the procedure of Example 4A, but replacing6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dionewith other compounds of formula (7), other compounds of formula (8) areprepared.

Example 5 Preparation of a Compound of Formula (1) A. Preparation of aCompound of Formula (1) in which R¹ is n-Propyl and R² is Ethyl

To a solution of6-amino-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dione (1.4 g, 7.1mmol) in a mixture of 50% acetic acid/water (35 ml) was added sodiumnitrite (2 g, 28.4 mmol) in portions over a period of 10 minutes. Themixture was stirred at 70° C. for 1 hour, then the reaction mixtureconcentrated to a low volume under reduced pressure. The solid wasfiltered off, and washed with water, to provide6-amino-1-ethyl-5-nitroso-3-propyl-1,3-dihydropyrimidine-2,4-dione, acompound of formula (1).

MS m/z 227.05 (M⁺), 249.08 (M⁺+Na)

B. Preparation of a Compound of Formula (1), Varying R¹ and R²

Similarly, following the procedure of Example 5A, but replacing6-amino-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dione with othercompounds of formula (8), the following compounds of formula (1) wereprepared:

-   6-amino-1-methyl-5-nitroso-3-propyl-1,3-dihydropyrimidine-2,4-dione;-   6-amino-1    ethyl-3-cyclopropylmethyl-5-nitroso-1,3-dihydropyrimidine-2,4-dione;-   6-amino-1-ethyl-3-cyclopropylmethyl-5-nitroso-1,3-dihydropyrimidine-2,4-dione;-   6-amino-1-methyl-3-(2-methylpropyl)-5-nitroso-1,3-dihydropyrimidine-2,4-dione;    and-   6-amino-1-ethyl-3-(2-methylpropy)-5-nitroso-1,3-dihydropyrimidine-2,4-dione.

C. Preparation of a Compound of Formula (1) Varying R¹ and R²

Similarly, following the procedure of Example 5A, but replacing6-amino-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dione with othercompounds of formula (8), other compounds of formula (1) are prepared.

Example 6 Preparation of a Compound of Formula (2) A. Preparation of aCompound of Formula (2) in which R¹ is n-Propyl and R² is Ethyl

To a solution of6-amino-1-ethyl-5-nitroso-3-propyl-1,3-dihydropyrimidine-2,4-dione (300mg) in methanol (10 ml) was added 10% palladium on carbon catalyst (50mg), and the mixture was hydrogenated under hydrogen at 30 psi for 2hours. The mixture was filtered through celite, and solvent was removedfrom the filtrate under reduced pressure, to provide5,6-diamino-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dione, a compoundof formula (2).

MS m/z 213.03 (M⁺), 235.06 (M⁺+Na)

B. Preparation of a Compound of Formula (2), Varying R¹ and R²

Similarly, following the procedure of Example 6A, but replacing6-amino-1-ethyl-5-nitroso-3-propyl-1,3-dihydropyrimidine-2,4-dione withother compounds of formula (1), the following compounds of formula (2)were prepared:

-   5,6-diamino-1-methyl-3-propyl-1,3-dihydropyrimidine-2,4-dione;-   5,6-diamino-1-methyl-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4-dione;-   5,6-diamino-1-ethyl-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4-dione;-   5,6-amino-1-methyl-3-(2-methylpropyl)-1,3-dihydropyrimidine-2,4-dione;    and-   5,6-diamino-1-ethyl-3-(2-methylpropyl)-1,3-dihydropyrimidine-2,4-dione.

C. Preparation of a Compound of Formula (2) Varying R¹ and R²

Similarly, following the procedure of Example 6A, but replacing6-amino-1-ethyl-5-nitroso-3-propyl-1,3-dihydropyrimidine-2,4-dione withother compounds of formula (1), other compounds of formula (2) areprepared.

Example 7 Preparation of a Compound of Formula (3) A. Preparation of aCompound of Formula (3) in which R¹ is n-Propyl, R² is Ethyl, X is1,4-Pyrazolyl, Y is Methylene, and Z is 3-Trifluoromethylphenyl

To a mixture of5,6-diamino-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dione (100 mg,0.47 mmol) and1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylic acid (0.151g, 0.56 mmol) in methanol (10 ml) was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.135 g,0.7 mmol), and the reaction mixture was stirred overnight at roomtemperature. The solvent was removed under reduced pressure, and theresidue purified using Bistag, eluting with 10% methanol/methylenechloride, to provideN-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}-pyrazol-4-yl)carboxamide.

¹H-NMR (DMSO-d6) δ 8.59 (s, 1H), 8.02 (s, 1H), 7.59-7.71 (m, 4H), 6.71(s, 2H), 5.51 (s, 2H), 3.91-3.96 (m, 2H), 3.70-3.75 (m, 2H), 1.47-1.55(m, 2H), 1.14 (t, 3H, J=7.03 Hz), 0.85 (t, 3H, J=7.42 Hz).

B. Preparation of a Compound of Formula (3), Varying R¹, R², X, Y, and Z

Similarly, following the procedure of Example 7A or 7B, but optionallyreplacing 5,6-diamino-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dionewith other compounds of formula (2), and optionally replacing1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylic acid withother compounds of formula Z—Y—X—CO₂H, the following compounds offormula (3) were prepared:

-   N-(6-amino-1-methyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}-pyrazol-4-yl)carboxamide;-   N-(6-amino-1-methyl-2,4-dioxo-3-cyclopropylmethyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}-pyrazol-4-yl)carboxamide;-   N-(6-amino-1-ethyl-2,4-dioxo-3-cyclopropylmethyl    (1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}-pyrazol-4-yl)carboxamide;-   N-(6-amino-1-methyl-2,4-dioxo-3-ethyl(1,3-dihydropyrimidin-5-yl))(1-{[3-fluorophenyl]methyl}-pyrazol-4-yl)carboxamide;-   N-(6-amino-1-methyl-2,4-dioxo-3-cyclopropylmethyl(1,3-dihydropyrimidin-5-yl))(1-{[3-fluorophenyl]methyl}-pyrazol-4-yl)carboxamide;-   N-(6-amino-1-ethyl-2,4-dioxo-3-cyclopropylmethyl(1,3-dihydropyrimidin-5-yl))(1-{[3-fluorophenyl]methyl}-pyrazol-4-yl)carboxamide;-   N-[6-amino-3-(cyclopropylmethyl)-1-methyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)][1-benzylpyrazol-4-yl]carboxamide;-   N-(6-amino-1-methyl-2,4-dioxo-3-cyclopropylmethyl(1,3-dihydropyrimidin-5-yl))(1-{[3-cyanophenyl]methyl}-pyrazol-4-yl)carboxamide;-   [1-(2-(1H-1,2,3,4-tetraazol-5-yl)ethyl)pyrazol-4-yl]-N-[6-amino-3-(cyclopropylmethyl)-1-methyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)]carboxamide;-   N-[6-amino-3-(cyclopropylmethyl)-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)](1-{[6-(trifluoromethyl)(3-pyridyl)]methyl}pyrazol-4-yl)carboxamide;-   N-[6-amino-3-propyl)-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)](1-{(2-pyridyl)]methyl}pyrazol-4-yl)carboxamide;-   N-[6-amino-3-(2-methylpropyl)-1-methyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)][1-benzylpyrazol-4-yl]carboxamide;-   N-[6-amino-3-(2-methylpropyl)-1-methyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)][1-{[3-fluorophenyl]methyl}pyrazol-4-yl]carboxamide;-   N-[6-amino-3-(2-methylpropyl)-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)][1-{[3-fluorophenyl]methyl}pyrazol-4-yl]carboxamide;-   N-[6-amino-3-(2-methylpropyl)-1-methyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)][1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl]carboxamide;    and-   N-[6-amino-3-(2-methylpropyl)-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)](1-{[6-(trifluoromethyl)(3-pyridyl)]methyl}pyrazol-4-yl)carboxamide.

C. Preparation of a Compound of Formula (2) Varying R¹ and R²

Similarly, following the procedure of Example 7A, but optionallyreplacing 5,6-diamino-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dionewith other compounds of formula (2), and optionally replacing1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylic acid withother compounds of formula Z—Y—X—CO₂H, other compounds of formula (3)are prepared.

Example 8 Preparation of a Compound of Formula I A. Preparation of aCompound of Formula I in which R¹ is n-Propyl, R² is Ethyl, X is1,4-Pyrazolyl, Y is Methylene, and Z is 3-Trifluoromethylphenyl

A mixture ofN-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-3-yl)carboxamide(80 mg, 0.17 mmol), 10% aqueous sodium hydroxide (5 ml), and methanol (5ml) was stirred at 100° C. for 2 hours. The mixture was cooled, methanolremoved under reduced pressure, and the residue diluted with water andacidified with hydrochloric acid. The precipitate was filtered off,washed with water, then methanol, to provide3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione,a compound of Formula I.

¹H-NMR (DMSO-d6) δ 8.57 (s, 1H), 8.15 (s, 1H), 7.60-7.75 (m, 4H), 5.54(s, 2H), 4.05-4.50 (m, 2H), 3.87-3.91 (m, 2H), 1.55-1.64 (m, 2H), 1.25(t, 3H, J=7.03 Hz), 0.90 (t, 3H, J=7.42 Hz); MS m/z 447.2 (M⁺).

B. Preparation of a Compound of Formula I, Varying R¹, R², X, Y, and Z

Similarly, following the procedure of Example 8A, but replacingN-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]-methyl}pyrazol-3-yl)carboxamidewith other compounds of formula (3), the following compounds of FormulaI were prepared:

-   1-cyclopropylmethyl-3-methyl-8-[1-(phenylmethyl)pyrazol-4-yl]-1,3,7-trihydropurine-2,6-dione;-   1-cyclopropylmethyl-3-methyl-8-{1-[(3-trifluoromethylphenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   1-cyclopropylmethyl-3-ethyl-8-{1-[(3-trifluoromethylphenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   1-cyclopropylmethyl-3-methyl-8-{1-[(3-fluorophenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   1-cyclopropylmethyl-3-ethyl-8-{1-[(3-fluorophenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   1-cyclopropylmethyl-3-ethyl-8-(1-{[6-(trifluoromethyl)(3-pyridyl)]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione;-   3-({4-[1-(cyclopropylmethyl)-3-methyl-2,6-dioxo-1,3,7-trihydropurin-8-yl]pyrazolyl}methyl)benzenecarbonitrile;-   8-[1-(2-(1H-1,2,3,4-tetraazol-5-yl)ethyl)pyrazol-4-yl]-3-methyl-1-cyclopropylmethyl-1,3,7-trihydropurine-2,6-dione;-   1-(2-methylpropyl)-3-methyl-8-[1-benzylpyrazol-4-yl]-1,3,7-trihydropurine-2,6-dione;-   1-(2-methylpropyl)-3-ethyl-8-{1-[(3-fluorophenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   1-(2-methylpropyl)-3-methyl-8-{1-[(3-trifluoromethylphenyl)methyl]pyrazol-4-yl}-1,3,7-tihydropurine-2,6-dione;-   1-(2-methylpropyl)-3-methyl-8-{1-[(3-fluorophenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;-   3-ethyl-1-(2-methylpropyl)-8-(1-{[6-(trifluoromethyl)(3-pyridyl)]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione;-   1-ethyl-3-methyl-8-{1-[(3-fluorophenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropurine-2,6-dione;    and-   3-ethyl-1-propyl-8-[1-(2-pyridylmethyl)pyrazol-4-yl]-1,3,7-trihydropurine-2,6-dione.

C. Preparation of a Compound of Formula I, Varying R¹, R², X, Y, and Z

Similarly, following the procedure of Example 8A, but replacingN-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]-methyl}pyrazol-3-yl)carboxamidewith other compounds of formula (3), other compounds of Formula I areprepared.

Example 9 Preparation of a Compound of Formula (10) A. Preparation of aCompound of Formula (10) in which R¹ is n-Propyl

A mixture of 6-aminouracil (5.08 g, 40 mmol), hexamethyldisilazane (50ml), and ammonium sulfate (260 mg, 1.96 mmol) was refluxed for 12 hours.After cooling, the solid was filtered off, and solvent was removed fromthe filtrate under reduced pressure to provide the trimethylsilylatedderivative of 6-aminouracil.

The product was dissolved in toluene (1.5 ml), and iodopropane (7.8 ml,80 mmol) and heated in an oil bath at 120° C. for 2 hours. The reactionmixture was then cooled to 0° C., and saturated aqueous sodiumbicarbonate added slowly. The resulting precipitate was filtered off,and washed sequentially with water, toluene, and ether, to provide6-amino-3-propyl-1,3-dihydropyrimidine-2,4-dione, a compound of formula(10), which was used in the next reaction with no further purification.

¹H-NMR (DMSO-d6) δ 10.34 (s, 1H), 6.16 (s, 2H), 4.54 (s, 1H), 3.57-3.62(m, 2H), 1.41-1.51 (m, 2H), 0.80 (t, 3H, J=7.43 Hz).

B. Preparation of a Compound of Formula (10), Varying R¹

Similarly, following the procedure of Example 9A, but replacingiodopropane with other alkyl halides of formula R¹Hal, other compoundsof formula (10) are prepared, including:

-   6-amino-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4-dione; and-   6-amino-3-(2-methylpropyl)-1,3-dihydropyrimidine-2,4-dione.

Example 10 Preparation of a Compound of Formula (11) A. Preparation of aCompound of Formula (10) in which R¹ is n-Propyl

To a solution of 6-amino-3-propyl-1,3-dihydropyrimidine-2,4-dione (5.6g) in a mixture of 50% acetic acid/water (160 ml) at 70° C. was addedsodium nitrite (4.5 g) in portions over a period of 15 minutes. Themixture was stirred at 70° C. for 45 minutes, then the reaction mixtureconcentrated to a low volume under reduced pressure. The solid wasfiltered off, and washed with water, to provide6-amino-5-nitroso-3-propyl-1,3-dihydropyrimidine-2,4-dione, a compoundof formula (11).

¹H-NMR (DMSO-d6) δ 11.42 (s, 1H), 7.98 (s, 1H), 3.77-3.81 (m, 2H), 3.33(s, 1H), 1.55-1.64 (m, 2H), 0.89 (t, 31-1, J=7.43 Hz); MS m/z 198.78(M⁺), 220.78 (M⁺+Na)

B. Preparation of a Compound of Formula (11), Varying R¹

Similarly, following the procedure of Example 10A, but replacing6-amino-3-propyl-1,3-dihydropyrimidine-2,4-dione with other compounds offormula (10), other compounds of formula (11) are prepared, including:

-   6-amino-5-nitroso-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4-dione;    and-   6-amino-5-nitroso-3-(2-methylpropyl)-1,3-dihydropyrimidine-2,4-dione.

Example 11 Preparation of a Compound of Formula (12) A. Preparation of aCompound of Formula (12) in which R¹ is n-Propyl

To a solution of6-amino-5-nitroso-3-propyl-1,3-dihydropyrimidine-2,4-dione (5.4 g, 27mmol) in 12.5% aqueous ammonia (135 ml) at 70° C. was added sodiumdithionite (Na₂S₂O₄, 9.45 g, 54 mmol) in portions over 15 minutes, andthe mixture was stirred for 20 minutes. The solution was concentratedunder reduced pressure, cooled to 5° C., the precipitate filtered off,and washed with cold water, to provide5,6-diamino-3-propyl-1,3-dihydropyrimidine-2,4-dione, a compound offormula (12).

¹H-NMR (DMSO-d6) δ 0.81 (t, 3H, J=7.43 Hz), 1.43-1.52 (m, 2H), 3.63-3.67(m, 2H), 5.56 (s, 2H); MS m/z 184.95 (M⁺) 206.96 (M⁺+Na)

B. Preparation of a Compound of Formula (12), Varying R¹

Similarly, following the procedure of Example 11A, but replacing6-amino-3-propyl-1,3-dihydropyrimidine-2,4-dione with other compounds offormula (11), other compounds of formula (12) are prepared, including:

-   5,6-diamino-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4-dione; and-   5,6-diamino-3-(2-methylpropyl)-1,3-dihydropyrimidine-2,4-dione.

Example 12 Preparation of a Compound of Formula (13) A. Preparation of aCompound of Formula (13) in which R¹ is n-Propyl, X is 1,4-Pyrazolyl, Yis Methylene, and Z is 3-Trifluoromethylphenyl

To a mixture of 5,6-diamino-3-propyl-1,3-dihydropyrimidine-2,4-dione(2.3 g, 126 mmol) and1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylic acid (3.79 g,14 mmol) in methanol (50 ml) was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.67 g, 14mmol), and the reaction mixture was stirred for 3 days at roomtemperature (although less time is acceptable). The precipitate wasfiltered off, and was washed sequentially with water, and methanol. Theproduct was dried under vacuum to provideN-(6-amino-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)carboxamide,a compound of formula (13).

¹H-NMR (DMSO-d6) δ 10.44 (s, 1H), 8.56 (s, 1H), 8.37 (s, 1H), 8.00 (s,1H), 7.56-7.71 (m, 3H), 6.02 (s, 1H), 5.49 (s, 2H), 3.62-3.66 (m, 2H),1.44-1.53 (m, 2H), 0.82 (t, 3H, J=7.43 Hz); MS m/z 458.92 (M⁺+Na).

B. Alternative Preparation of a Compound of Formula (3) in which R¹ isn-Propyl, X is 1,4-Pyrazolyl, Y is Methylene, and Z is3-Trifluoromethylphenyl

A solution of 1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylicacid (1 g, 3.7 mmol) in thionyl chloride (1 ml) was heated at 70° C. for4 hours. Excess thionyl chloride was distilled off, and the residuetreated with methylene chloride/hexanes. The solvent was removed underreduced pressure, and the residue dissolved in acetonitrile. Thissolution was added to a suspension of5,6-diamino-3-propyl-1,3-dihydropyrimidine-2,4-dione (2.3 g, 126 mmol)and triethylamine (1 ml) in acetonitrile (20 ml) at 0° C., and stirredfor 16 hours. The reaction mixture was quenched with water (5 ml),acidified with hydrochloric acid, stirred for 30 minutes, and theprecipitate filtered off. The product was washed with ether, to provideN-(6-amino-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)carboxamide,a compound of formula (13).

C. Preparation of a Compound of Formula (13), Varying R¹, X, Y, and Z

Similarly, following the procedure of Example 12A or 12B, but optionallyreplacing 6-amino-3-propyl-1,3-dihydropyrimidine-2,4-dione with othercompounds of formula (12), and optionally replacing1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylic acid withother compounds of formula Z—Y—X—CO₂H, other compounds of formula (13)are prepared, including:

-   N-(6-amino-2,4-dioxo-3-cyclopropylmethyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)carboxamide;-   N-(6-amino-2,4-dioxo-3-(2-methylpropyl)(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)carboxamide;-   N-(6-amino-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-fluorophenyl]methyl}pyrazol-4-yl)carboxamide;-   N-(6-amino-2,4-dioxo-3-cyclopropylmethyl(1,3-dihydropyrimidin-5-yl))(1-{[3-fluorophenyl]methyl}pyrazol-4-yl)carboxamide;-   N-(6-amino-2,4-dioxo-3-(2-methylpropyl)(1,3-dihydropyrimidin-5-yl))(1-{[3-fluorophenyl]methyl}pyrazol-4-yl)carboxamide;-   N-(6-amino-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl)(1-[1-benzyl]pyrazol-4-yl)carboxamide;-   N-(6-amino-2,4-dioxo-3-cyclopropylmethyl(1,3-dihydropyrimidin-5-yl))(1-[1-benzyl]pyrazol-4-yl)carboxamide;-   N-(6-amino-2,4-dioxo-3-(2-methylpropyl)(1,3-dihydropyrimidin-5-yl))(1-[1-benzyl]pyrazol-4-yl)carboxamide;-   N-(6-amino-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-cyanophenyl]methyl}pyrazol-4-yl)carboxamide;-   N-(6-amino-2,4-dioxo-3-cyclopropylmethyl(1,3-dihydropyrimidin-5-yl))(1-{[3-cyanophenyl]methyl}pyrazol-4-yl)carboxamide;-   N-(6-amino-2,4-dioxo-3-(2-methylpropyl)(1,3-dihydropyrimidin-5-yl))(1-{[3-cyanophenyl]methyl}pyrazol-4-yl)carboxamide;-   N-(6-amino-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-[1-(2-(1H-1,2,3,4-tetraazol-5-yl)ethyl)pyrazol-4-yl]carboxamide;-   N-(6-amino-2,4-dioxo-3-cyclopropylmethyl    (1,3-dihydropyrimidin-5-yl))(1-{[1-(2-(1H-1,2,3,4-tetraazol-5-yl)ethyl)pyrazol-4-yl)carboxamide;-   N-(6-amino-2,4-dioxo-3-(2-methylpropyl)(1,3-dihydropyrimidin-5-yl))(1-{[1-(2-(1H-1,2,3,4-tetraazol-5-yl)ethyl)pyrazol-4-yl)carboxamide;-   N-(6-amino-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[6-(trifluoromethyl)(3-pyridyl)]-methyl}pyrazol-4-yl)carboxamide;-   N-(6-amino-2,4-dioxo-3-cyclopropylmethyl(1,3-dihydropyrimidin-5-yl))(1-{[6-(trifluoromethyl)(3-pyridyl)]methyl}pyrazol-4-yl)carboxamide;    and-   N-(6-amino-2,4-dioxo-3-(2-methylpropyl)(1,3-dihydropyrimidin-5-yl))(1-{[6-(trifluoromethyl)(3-pyridyl)]methyl}pyrazol-4-yl)carboxamide.

Example 13 Preparation of a Compound of Formula (3) A. Preparation of aCompound of Formula (3) in which R¹ is n-Propyl, R² is Ethyl, X is1,4-Pyrazolyl, Y is Methylene, and Z is 3-Trifluoromethylphenyl

A mixture of a solution ofN-(6-amino-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)-phenyl]methyl}pyrazol-3-yl)carboxamide(872 mg, 2 mmol) in dimethylformamide (10 ml), potassium carbonate (552mg, 4 mmol) and ethyl iodide (0.24 ml, 3 mmol) was stirred at roomtemperature overnight. The reaction mixture was filtered, and thesolvent was evaporated from the filtrate under reduced pressure. Theresidue was stirred with water for two hours at room temperature, andthe precipitate filtered off, washed with water, and then dissolved inmethanol. The solvent was then removed under reduced pressure to provideN-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)carboxamide,a compound of formula (3).

¹H-NMR (DMSO-d6): δ 8.58 (s, 1H), 8.39 (s, 1H), 8.01 (s, 1H), 7.72-7.50(m, 4H), 6.71 (s, 2H), 5.51 (s, 2H), 4.0-3.82 (m, 2H), 3.77-3.65 (m,2H), 1.60-1.50 (m, 2H), 1.13 (t, 3H, J=6.8 Hz), 0.84 (t, 3H, J=7.2 Hz);MS m/z 462.9 (M⁻)

B. Preparation of a Compound of Formula (13), Varying R¹, X, Y, and Z

Similarly, following the procedure of Example 13A, but replacingN-(6-amino-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)-1-phenyl]methyl}pyrazol-3-yl)carboxamidewith other compounds of formula (13), other compounds of formula (3) areprepared, including:

-   N-(6-amino-1-methyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}-pyrazol-4-yl)carboxamide;-   N-(6-amino-1-methyl-2,4-dioxo-3-cyclopropylmethyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}-pyrazol-4-yl)carboxamide;-   N-(6-amino-1-ethyl-2,4-dioxo-3-cyclopropylmethyl    (1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}-pyrazol-4-yl)carboxamide;-   N-(6-amino-1-methyl-2,4-dioxo-3-ethyl(1,3-dihydropyrimidin-5-yl))(1-{[3-fluorophenyl]methyl}-pyrazol-4-yl)carboxamide;-   N-(6-amino-1-methyl-2,4-dioxo-3-cyclopropylmethyl(1,3-dihydropyrimidin-5-yl))(1-{[3-fluorophenyl]methyl}-pyrazol-4-yl)carboxamide;-   N-(6-amino-1-ethyl-2,4-dioxo-3-cyclopropylmethyl(1,3-dihydropyrimidin-5-yl))(1-{[3-fluorophenyl]methyl}-pyrazol-4-yl)carboxamide;-   N-[6-amino-3-(cyclopropylmethyl)-1-methyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)][1-benzylpyrazol-4-yl]carboxamide;-   N-(6-amino-1-methyl-2,4-dioxo-3-cyclopropylmethyl(1,3-dihydropyrimidin-5-yl))(1-{[3-cyanophenyl]methyl}-pyrazol-4-yl)carboxamide;-   [1-(2-(1H-1,2,3,4-tetraazol-5-yl)ethyl)pyrazol-4-yl]-N-[6-amino-3-(cyclopropylmethyl)-1-methyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)]carboxamide;-   N-[6-amino-3-(cyclopropylmethyl)-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)](1-{[6-(trifluoromethyl)(3-pyridyl)]methyl}pyrazol-4-yl)carboxamide;-   N-[6-amino-3-propyl)-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)](1-{(2-pyridyl)]methyl}pyrazol-4-yl)carboxamide;-   N-[6-amino-3-(2-methylpropyl)-1-methyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)][1-benzylpyrazol-4-yl]carboxamide;-   N-[6-amino-3-(2-methylpropyl)-1-methyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)][1-{[3-fluorophenyl]methyl}pyrazol-4-yl]carboxamide;-   N-[6-amino-3-(2-methylpropyl)-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)][1-{[3-fluorophenyl]methyl}pyrazol-4-yl]carboxamide;-   N-[6-amino-3-(2-methylpropyl)-1-methyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)][1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl]carboxamide;    and-   N-[6-amino-3-(2-methylpropyl)-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)](1-{[6-(trifluoromethyl)(3-pyridyl)]methyl}pyrazol-4-yl)carboxamide.

Example 14 Preparation of a Compound of Formula I A. Preparation of aCompound of Formula I in which R¹ is n-Propyl, R² is Ethyl, X is1,4-Pyrazolyl, Y is Methylene, and Z is 3-Trifluoromethylphenyl

A mixture ofN-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-3-yl)carboxamide(850 mg, 2.34 mmol), 10% aqueous sodium hydroxide (10 ml), and methanol(10 ml) was stirred at 100° C. for 18 hours. The mixture was cooled,methanol removed under reduced pressure, and the remaining mixture wasacidified with hydrochloric acid to pH 2. The precipitate was filteredoff, washed with water/methanol mixture, to provide3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione,a compound of Formula I.

¹H-NMR (DMSO-d6) δ 8.57 (s, 1H), 8.15 (s, 1H), 7.60-7.75 (m, 4H), 5.54(s, 2H), 4.05-4.50 (m, 2H), 3.87-3.91 (m, 2H), 1.55-1.64 (m, 2H), 1.25(t, 3H, J=7.03 Hz), 0.90 (t, 3H, J=7.42 Hz); MS m/r 447.2 (M⁺)

B. Preparation of a Compound of Formula I, Varying R¹, R², X, Y, and Z

Similarly, following the procedure of Example 14A, but replacingN-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-3-yl)carboxamidewith other compounds of formula (13), other compounds of Formula I areprepared, including those listed in Example 8.

Example 15 Preparation of a Compound of Formula (14) A. Preparation of aCompound of Formula (14) in which R² is Ethyl

To a solution of 6-amino-1-ethyl-1,3-dihydropyrimidine-2,4-dione (5.0 g,32.3 mmol) in a mixture of 50% acetic acid/water (50 ml) at 70° C. wasadded sodium nitrite (4.45 g, 64.5 mmol) in portions over a period of 30minutes. The mixture was stirred at 70° C. for a further 30 minutes. Thereaction mixture was cooled, and the precipitate filtered off, andwashed with water, then methanol, to provide6-amino-1-ethyl-5-nitroso-1,3-dihydropyrimidine-2,4-dione, a compound offormula (14).

¹H-NMR (DMSO-d6): δ 11.52 (s, 1H), 9.16 (s, 1H), 3.83 (q, 2H, J=7.0 Hz),1.11 (t, 3H, J=7.0 Hz). MS m/z 184.8 (M⁺), 206.80 (M⁺+Na)

B. Preparation of a Compound of Formula (14), Varying R²

Similarly, following the procedure of Example 15A, but replacing6-amino-1-ethyl-1,3-dihydropyrimidine-2,4-dione with6-amino-1-methyl-1,3-dihydropyrimidine-2,4-dione,6-amino-1-methyl-5-nitroso-1,3-dihydropyrimidine-2,4-dione was prepared.

C. Preparation of a Compound of Formula (14), Varying R²

Similarly, following the procedure of Example 15A, but replacing6-amino-1-ethyl-1,3-dihydropyrimidine-2,4-dione with other compounds offormula (5), other compounds of formula (14) are prepared.

Example 16 Preparation of a Compound of Formula (15) A. Preparation of aCompound of Formula (15) in which R² is Ethyl

To a solution of6-amino-1-ethyl-5-nitroso-1,3-dihydropyrimidine-2,4-dione (3.9 g, 21.2mmol) in 14.5% aqueous ammonia (50 ml) at 50° C. was added sodiumdithionite (Na₂S₂O₄, 7.37 g, 42.4 mmol) in portions over 15 minutes, andthe mixture was stirred for 20 minutes. The solution was concentratedunder reduced pressure to a volume of 30 ml, cooled to 5° C., theprecipitate filtered off, and washed with cold water, to provide5,6-diamino-1-ethyl-1,3-dihydropyrimidine-2,4-dione, a compound offormula (15).

¹H-NMR (DMSO-d6): δ 10.58 (s, 1H), 6.18 (s, 2H), 3.83 (q, 2H, J=7.2 Hz),2.82 (s, 2H), 1.10 (t, 3H, J=7.2 Hz).

B. Preparation of a Compound of Formula (15), Varying R²

Similarly, following the procedure of Example 16A, but replacing6-amino-1-ethyl-5-nitroso-1,3-dihydropyrimidine-2,4-dione with6-amino-1-methyl-5-nitroso-1,3-dihydropyrimidine-2,4-dione,5,6-diamino-1-methyl-1,3-dihydropyrimidine-2,4-dione was prepared.

C. Preparation of a Compound of Formula (15), Varying R²

Similarly, following the procedure of Example 16A, but replacing6-amino-1-ethyl-5-nitroso-1,3-dihydropyrimidine-2,4-dione with othercompounds of formula (14), other compounds of formula (15) are prepared.

Example 17 Preparation of a Compound of Formula (16) A. Preparation of aCompound of Formula (16) in which R² is Ethyl, X is 1,4-Pyrazolyl, Y isMethylene, and Z is 3-Trifluoromethylphenyl

To a mixture of 5,6-diamino-1-ethyl-1,3-dihydropyrimidine-2,4-dione (2g, 11.76 mmol) and1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylic acid (3.5 g,12.94 mmol) in methanol (50 ml) was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.47 g,12.94 mmol), and the reaction mixture was stirred for 16 hours at roomtemperature. Solvent was removed under reduced pressure, and the residuewas washed with water and methanol. The product was dried under vacuumto provideN-(6-amino-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)carboxamide,a compound of formula (16).

¹H-NMR (DMSO-d6): δ 10.60 (s, 1H), 8.50 (s, 1H), 8.39 (s, 1H), 8.01 (s,1H), 7.72-7.50 (m, 4H), 6.69 (s, 2H), 5.50 (s, 2H), 3.87 (q, 2H, J=7.2Hz), 1.1.1 (t, 3H, 7.2 Hz); MS m/z 421 (M⁻)

B. Preparation of a Compound of Formula (16), Varying R², X, Y, and Z

Similarly, following the procedure of Example 17A, but replacing5,6-diamino-1-ethyl-1,3-dihydropyrimidine-2,4-dione with5,6-diamino-1-methyl-1,3-dihydropyrimidine-2,4-dione,N-(6-amino-1-methyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)carboxamidewas prepared.

C. Preparation of a Compound of Formula (16), Varying R², X, Y, and Z

Similarly, following the procedure of Example 16A, but replacing5,6-diamino-1-ethyl-1,3-dihydropyrimidine-2,4-dione with other compoundsof formula (14), other compounds of formula (15) are prepared.

Example 18 Preparation of a Compound of Formula (3) A. Preparation of aCompound of Formula (3) in which R¹ is n-Propyl, R² is Ethyl, X is1,4-Pyrazolyl, Y is Methylene, and Z is 3-Trifluoromethylphenyl

A mixture of a solution ofN-(6-amino-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-3-yl)carboxamide(1.5 g, 3.55 mmol) in dimethylformamide (30 ml), potassium carbonate(980 mg, 7.1 mmol) and propyl iodide (724 mg, 4.26 mmol) was stirred atroom temperature overnight. Water was added, and the precipitatefiltered off, to provideN-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)carboxamide,a compound of formula (3), which was used in the next reaction with nofurther purification.

¹H-NMR (DMSO-d6): δ 8.58 (s, 1H), 8.39 (s, 1H), 8.01 (s, 1H), 7.72-7.50(m, 4H), 6.71 (s, 2H), 5.51 (s, 2H), 4.0-3.82 (m, 2H), 3.77-3.65 (m,2H), 1.60-1.50 (m, 2H), 1.13 (t, 3H, J=6.8 Hz), 0.84 (t, 3H, J=7.2 Hz);MS m/z 462.9 (M⁻)

B. Preparation of a Compound of Formula (3), Varying R¹, R², X, Y, and Z

Similarly, following the procedure of Example 18A, but replacingN-(6-amino-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]-methyl}pyrazol-3-yl)carboxamidewith N-(6-amino-1-methyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)),N-(6-amino-1-methyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)carboxamidewas prepared.

C. Preparation of a Compound of Formula (3), Varying R¹, R², X, Y, and Z

Similarly, following the procedure of Example 18A, but optionallyreplacingN-(6-amino-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-3-yl)carboxamidewith other compounds of formula (15), and optionally replacing propyliodide with other compounds of formula R¹Hal, other compounds of formula(3) are prepared.

Example 19 Preparation of a Compound of Formula I A. Preparation of aCompound of Formula I in which R¹ is n-Propyl, R² is Ethyl, X is1,4-Pyrazolyl, Y is Methylene, and Z is 3-Trifluoromethylphenyl

A mixture ofN-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-3-yl)carboxamide(300 mg, 464 mmol), 20% aqueous sodium hydroxide (5 ml), and methanol(10 ml) was stirred at 80° C. for 3 hours. The mixture was cooled,methanol removed under reduced pressure, and the remaining mixture wasacidified with hydrochloric acid to pH 2. The precipitate was filteredoff, washed with water and methanol, to provide3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione,a compound of Formula I.

¹H-NMR (DMSO-d6) δ 8.57 (s, 1H), 8.15 (s, 1H), 7.60-7.75 (m, 4H), 5.54(s, 2H), 4.05-4.50 (m, 2H), 3.87-3.91 (m, 2H), 1.55-1.64 (m, 2H), 1.25(t, 3H, J=7.03 Hz), 0.90 (t, 3H, J=7.42 Hz); MS m/z 447.2 (M⁺)

Example 20 Characterization of A_(2B) Antagonists

Radioligand Binding for A_(2B) Adenosine Receptor

Human A_(2B) adenosine receptor cDNA was stably transfected into HEK-293cells (referred to as HEK-A2B cells). Monolayers of HEK-A2B cells werewashed with PBS once and harvested in a buffer containing 10 mM HEPES(pH 7.4), 10 mM EDTA and protease inhibitors. These cells werehomogenized in polytron for 1 minute at setting 4 and centrifuged at29000 g for 15 minutes at 4° C. The cell pellets were washed once with abuffer containing 10 mM HEPES (pH7.4), 1 mM EDTA and proteaseinhibitors, and were resuspended in the same buffer supplemented with10% sucrose. Frozen aliquots were kept at −80° C.

Competition assays were started by mixing 10 nM ³H-ZM241385 (TocrisCookson) with various concentrations of test compounds and 50 μgmembrane proteins in TE buffer (50 mM Tris and 1 mM EDTA) supplementedwith 1 Unit/mL adenosine deaminase. The assays were incubated for 90minutes, stopped by filtration using Packard Harvester and washed fourtimes with ice-cold TM buffer (10 mM Tris, 1 mM MgCl2, pH 7.4). Nonspecific binding was determined in the presence of 10 μM ZM241385. Theaffinities of compounds (i.e. Ki values) were calculated using GraphPadsoftware.

Radioligand Binding for Other Adenosine Receptors

Human A₁, A_(2A), A₃ adenosine receptor cDNAs were stably transfectedinto either CHO or HEK-293 cells (referred to as CHO-A₁, HEK-A_(2A),CHO-A₃). Membranes were prepared from these cells using the sameprotocol as described above. Competition assays were started by mixing0.5 nM ³H-CPX (for CHO-A₁), 2 nM ³H-ZM214385 (HEK-A_(2A)) or 0.1 nM¹²⁵I-AB-MECA (CHO-A₃) with various concentrations of test compounds andthe perspective membranes in TE buffer (50 mM Tris and 1 mM EDTA ofCHO-A₁ and HEK-A_(2A)) or TEM buffer (50 mM Tris, 1 mM EDTA and 10 mMMgCl₂ for CHO-A₃) supplemented with 1 Unit/mL adenosine deaminase. Theassays were incubated for 90 minutes, stopped by filtration usingPackard Harvester and washed four times with ice-cold TM buffer (10 mMTris, 1 mM MgCl₂, pH 7.4). Non specific binding was determined in thepresence of 1 μM CPX (CHO-A₁), 1 μM ZM241385 (HEK-A_(2A)) and 1 μMIB-MECA (CHO-A₃). The affinities of compounds (i.e. Ki values) werecalculated using GraphPad™ software.

cAMP Measurements

Monolayer of transfected cells were collected in PBS containing 5 mMEDTA. Cells were washed once with DMEM and resuspended in DMEMcontaining 1 Unit/mL adenosine deaminase at a density of 100,000-500,000cells/ml. 100 μl of the cell suspension was mixed with 25 μl containingvarious agonists and/or antagonists and the reaction was kept at 37° C.for 15 minutes. At the end of 15 minutes, 125 μl 0.2N HCl was added tostop the reaction. Cells were centrifuged for 10 minutes at 1000 rpm.100 μl of the supernatant was removed and acetylated. The concentrationsof cAMP in the supernatants were measured using the direct cAMP assayfrom Assay Design. A_(2A) and A_(2B) adenosine receptors are coupled toGs proteins and thus agonists for A_(2A), adenosine receptor (such asCGS21680) or for A_(2B) adenosine receptor (such as NECA) increase thecAMP accumulations whereas the antagonists to these receptors preventthe increase in cAMP accumulations-induced by the agonists. A₁ and A₃adenosine receptors are coupled to Gi proteins and thus agonists for A₁adenosine receptor (such as CPA) or for A₃ adenosine receptor (such asIB-MECA) inhibit the increase in cAMP accumulations-induced byforskolin. Antagonists to A₁ and A₃ receptors prevent the inhibition incAMP accumulations.

Real-time RT-PCR was performed to determine the expression levels of theadenosine receptor (AdoR) subtypes on primary cultured human hepaticstellate cells (HHSCs). Among the four subtypes of AdoRs, the A_(2B)AdoR was expressed at the highest level. In addition, using cellularcAMP concentration as a functional readout, our results indicated thatA_(2B) AdoRs are functionally expressed on HHSCs, whereas A₁, A_(2A), orA₃ AdoRs are not. The effect of adenosine or NECA, a stable analog ofadenosine, on the expression of the inflammatory cytokines wasdetermined using ELISA. Adenosine and NECA increased the release of IL-6in a concentration-dependent manner with a maximal increase of 11.9±3.1fold over the basal level. In addition, NECA increased the expression ofα-smooth muscle actin and α-1 pro-collagen and the production ofcollagen from HHSCs. The effects of NECA were completely abolished bythe A_(2B) AdoR antagonist and partially blocked by an IL-6 neutralizingantibody.

Example 21 Effect of A) Antagonist on Human Hepatic Stellate Cells

Abbreviations Ab Antibody AdoR Adenosine receptor ADA adenosinedeaminase ANOVA analysis of variance AST aspartate aminotransferae CPAN6-cyclopentyladenosine DMEM Dulbecco's modified Eagle's medium DMSOdimethyl sulfoxide DNase deoxyribonuclease HEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HHSCs human hepaticstellate cells NECA 5′-(N-ethylcarboxamido)-adenosine

Materials and Methods

Materials

Selective antagonists to the A_(2B) receptor(8-(1-{[5-(4-chlorophenyl)(1,2,4-oxadiazol-3-yl)]methyl}pyrazol-4-yl)-1-propyl-1,3,7-trihydropurine-2,6-dione(compound (1)) and3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione(compound (2)) were synthesized by the Department of Bio-OrganicChemistry at CV Therapeutics Inc. (Palo Alto, Calif.), and was describedin our earlier publication Zhong et al. (2004) Am J Respir Cell MolBiol; 30(1):118-125. All other reagents, such as rolipram, forskolin,NECA, and adenosine deaminase (ADA), etc., were purchased from Sigma(St. Louis, Mo.) unless otherwise stated.

Cell Culture

Primary cultured normal human hepatic stellate Cells (HHSCs) wereobtained from ScienCell Research Laboratories (San Diego, Calif.) andcultured using stellate cell growth medium (ScienCell ResearchLaboratories). HHSCs were routinely grown in a humidified incubator with5% CO₂ at 37° C. and reseeded when they reached about 80-90% confluency.Each re-seeding is called a passage. Cells from passages 2 to 5 wereused in the following studies.

Stimulation of HHSCs

HHSCs were seeded into 12-well tissue culture plates at a density of1×10⁵ cells/well in stellate cell growth medium and allowed to adhereovernight and reach ˜90% confluency. Cells were washed twice in HEPESbuffered saline, and cultured in DMEM containing various agonists orantagonists of AdoRs for 1 or 24 h.

RNA Extraction and Real-Time RT-PCR

Total RNA was extracted from HHSCs using the Stratagene Absolutely RNA™RT-PCR Miniprep Kit followed by DNase treatment to eliminate potentialgenomic DNA contamination. Real-time RT-RCR for adenosine receptors wasperformed as previously described in Zhong et al. (2004) Am J RespirCell Mol Biol; 30(1):118-125.

The specific primers for α-smooth muscle actin used were:

forward: 5′ TGGGAATGGGACAAAAAGACA3′; (SEQ ID NO.: 1) and reverse: 5′CGGGTACTTCAGGGTCAGGAT3′, (SEQ ID NO.: 2)while the primers for α-1 pro-collagen were:

forward: 5′ CACCAATCACCTGCGTACAGA3′; (SEQ ID NO.: 3) and reverse: 5′CAGATCACGTCATCGCACAAC3′, (SEQ ID NO.: 4)and each of these primers were designed using Primer Express 2.0(Applied Biosystems) following the recommended guidelines based onsequences from Genbank. At the end of the PCR cycle, a dissociationcurve was generated to ensure the amplification of a single product, andthe threshold cycle time (Ct value) for each gene was determined.Relative mRNA levels were calculated based on the Ct values, normalizedto β-actin in the same sample, and presented as percentages of β-actinmRNA.Measurement of cAMP Accumulation

Cells were harvested using 0.0025% trypsin and 2 mM EDTA in PBS, washedand resuspended in phenol-free DMEM to a concentration of 1×10⁶cells/ml, and then incubated with 1 U/ml of ADA for 30 min at roomtemperature. Cells were then treated with AdoR agonists, antagonists,and forskolin in the presence of 50 μM of the phosphodiesterase IVinhibitor, rolipram. After incubating for 15 min at 37° C., cells werelysed and cAMP concentrations were determined using cAMP-Screen Direct™System (Applied Biosystems) according to the manufacturer'sinstructions.

Measurement of IL-6, Collagen and Aspartate Aminotransferae (AST)

The concentration of IL-6 in the cell medium was determined using ELISAkits obtained from Biosource (Camarillo, Calif.) according to themanufacturer's instructions. The minimal detection levels of IL-6 withthese kits were 2 pg/ml. The concentration of soluble collagen in thecell medium was measured using the Sircol collagen assay (Biocolor Ltd.,Belfast N. Ireland) according to manufacture's instructions. Theactivity of AST in mouse plasma was determined using the Infinity™ ASTassay (Thermo Electron Corporation, Waltham, Mass.)

Mice

Adenosine deaminase (ADA)-deficient mice were generated and genotyped asdescribed in Blackburn et al. (1998), J Biol Chem, 273(9):5093-5100.Mice homozygous for the null Ada allele were designated ADA-deficient(ADA^(−/−)), while mice heterozygous for the null Ada allele weredesignated as ADA control mice (ADA⁺). All mice were on a mixed129sv/C57BL/6J background and all phenotypic comparisons were performedamongst littermates. Animal care was in accordance with institutionaland NIH guidelines. Mice were housed in ventilated cages equipped withmicroisolator lids and maintained under strict containment protocols. Noevidence of bacterial, parasitic, or fungal infection was found, andserologies on cage littermates were negative for 12 of the most commonmurine viruses.

Antagonist Treatment

ADA^(−/−) mice were identified at birth by screening for ADA enzymaticactivity in the blood as described by Young et al. (2004) J Immunol,173(2):1380-1389. ADA^(−/−) mice were maintained on ADA enzyme therapyfrom postnatal day 2 until postnatal day 21 also as described in Younget al. (2004). At this stage, treatments with compound (2) (1 mg/kg perinjection) or vehicle (corn oil/ethanol/DMSO) were initiated. Treatmentsconsisted of an intra-peritoneal i.p. injection in the morning and inthe evening for 17 days. Treatment groups included ADA^(−/−) or ADA⁺mice receiving compound (2), vehicle or no treatment. All mice werelittermates and both males and females were included in theseexperiments.

Statistical Analysis

Data were presented as mean±SEM of at least three separate experiments.The statistical analysis was performed using a two-tailed Student'st-test, or ANOVA followed by Newman-Keuls test for multiple comparisons.A p value of <0.05 was considered significant.

Results

Expression of AdoR Subtypes in HHSCs

Real-time RT-PCR was performed to quantify the levels of transcripts forAdoRs. Among the four subtypes, the A_(2B) receptor had the highesttranscript level (FIG. 1). Lower levels of A₁ and A_(2A) receptortranscripts were also detected, whereas the transcript for A₃ receptorswas below the detection level. Hence, the rank order of AdoR mRNA levelswas A_(2B)>>A_(2A)>A₁>>A₃.

In many cell types, activation of A₂ or A_(2B) receptors leads toincreases in cellular cAMP accumulation, whereas activation of A₁ or A₃receptors decreases cellular cAMP accumulation caused by the adenylatecyclase activator, forskolin. To identify the AdoR subtype(s) that arefunctionally expressed in HHSCs, the effects of a non-selective agonistNECA and several other selective agonists on cellular cAMP accumulationwere determined. NECA is a stable analog of adenosine, and it activatesall four AdoR subtypes including A_(2B) receptors. As shown in FIG. 2A,NECA increased cellular cAMP accumulation in a concentration-dependentmanner. In contrast, the A_(2A) selective agonist CGS-21680 (=10 μM) didnot cause a significant increase in cellular cAMP concentration. Inaddition, the A₁ selective agonist, CPA (1 μM), and the A₃ selectiveagonist, IB-MECA (1 μM), failed to inhibit the cellular cAMPaccumulation caused by forskolin (10 μM, FIG. 2B).

Because there is no selective agonist for A_(2B) receptors, the effectof a selective antagonist to A_(2B) receptors, compound (1), onNECA-induced cellular cAMP accumulation was determined. Compound (1) hasa high affinity for the A_(2B) receptor (Ki=7 nM) and very low affinityfor three other AdoR subtypes (Ki values are more than 5 μM for A₁,A_(2A), and A₃ receptors) (Zhong et al. (2004) and Zhong et al. (2005)Am J Respir Cell Mol Biol, 32(1):2-8). As shown in FIG. 2A, compound (1)(1 μM) significantly attenuated NECA-induced cellular cAMP accumulation.Thus, using cellular cAMP concentration as readout for the functionalexpression of AdoRs, the results indicate that A_(2B) receptors arefunctionally expressed in HHSCs whereas A₁, A_(2A), or A₃ receptors arenot.

Activation of the A_(2B) Receptor Increased the Release of IL-6 fromHHSCs

The concentrations of IL-6 in the culture media from cells treated withadenosine and NECA were measured using ELISA. As shown in FIG. 3, bothadenosine and NECA increased IL-6 release in a concentration-dependentmanner. NECA (100 μM) caused 11.9±3.1 fold increase of IL-6 releasecompared to vehicle-treated cells. To determine the role of A_(2B)receptors in NECA-induced IL-6 production, cells were incubated withcompound (1) (1 μM) together with NECA (10 μM). The A_(2B) receptorantagonist, compound (1) (1 μM) reduced the NECA-increased IL-6 releaseby 90.7±0.1% (FIG. 3B). These results confirmed that NECA-induced IL-6release is mediated by the A_(2B) receptor subtype.

Effect of NECA on Expression of α-Smooth Muscle Actin and α-1Pro-Collagen

Activation of HHSC with accumulation of interstitial collagens is ahallmark of liver fibrosis. α-smooth muscle actin is a marker formyofibroblast and hence increased expression of α-smooth muscle actin isan indicator for HHSC differentiation into myofibroblast. The effect ofNECA on expression of α-smooth muscle actin and α-1 pro-collagen wasdetermined using real-time RT-PCR. As shown in FIG. 4, NECAsignificantly increased the expression of both α-smooth muscle actin(FIG. 4A) and α-1 pro-collagen (FIG. 4B). These results suggested thatNECA may promote HHSC activation and collagen production.

Increased Collagen Production by Activation of the A_(2B) Receptor isPartially Mediated by IL-6

To confirm the role of NECA in collagen production, the concentrationsof soluble collagen in the culture media from cells treated with NECAwere measured. NECA caused a significant increase in release of collagen(FIG. 5). This effect of NECA was abolished by A_(2B) antagonist,compound (1). To determine whether the effect of NECA on collagenproduction is dependent on the release of IL-6, the IL-6 neutralizing Abwas added to the cell media during NECA treatment. The IL-6 neutralizingAb partially and significantly decreased the effect of NECA. Theseresults demonstrate that activation of the A_(2B) receptor leads toincreased collagen production and this effect is partially mediated byreleased IL-6 from HHSCs.

Effect of A_(2B) Receptor Antagonism on AST Levels in ADA-Deficient Mice

ADA metabolizes adenosine; hence elevated adenosine levels arewidespread among the tissues, including the liver of ADA-deficient mice,see Blackburn et al. (1998), J Biol Chem, 273(9):5093-5100. The plasmaAST levels were examined in ADA⁺ and ADA^(−/−) mice treated with vehicleor compound (2). AST levels were elevated in ADA^(−/−) mice compared toADA⁺ mice. Treatment of ADA^(−/−) mice with compound (2) resulted in asignificant reduction in AST levels (FIG. 6). These results suggest thatA_(2B) receptor antagonism can prevent AST elevation in ADA^(−/−) mice.

We claim:
 1. A method of inhibiting hepatic fibrosis as a result ofliver replacement or repair in a human in need thereof by administeringa therapeutically effective amount of a compound having the name3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione,represented by the chemical formula

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the compound is administered prior to the liver replacement orrepair.
 3. The method of claim 1, wherein the compound is administeredconcurrently with the liver replacement or repair.
 4. The method ofclaim 1, wherein the compound is administered after the liverreplacement or repair.
 5. The method of claim 1, wherein the compound isadministered orally.
 6. The method of claim 1, wherein the compound isadministered by IV.